Methods for preparing pharmaceutical formulations

ABSTRACT

The invention relates to pharmaceutical formulations and methods for preparing pharmaceutical formulations of histamine releasers. The present invention provides methods for determining the concentration of physiologically acceptable excipient for use in the formulations of invention. The present invention also provides methods for suppressing pharmaceutically-induced histamine release by administering to an animal, the formulations of the present invention. A kit useful for preparing pharmaceutical formulations of histamine releasers is also provided.

BACKGROUND OF THE INVENTION

[0001] The present invention relates to the use of pharmaceuticallyactive agents known to cause histamine release when intravenouslyadministered to an animal. More particularly the present inventionrelates to new methods of using such pharmaceutically active agents andnew formulations of such agents which address pharmaceutically-inducedhistamine release.

[0002] The cardiovascular and respiratory effects indicative ofundesirable degrees of histamine release which are specific to someconventional pharmacological agents have been troubling clinicians fordecades. The clinical observations associated with an undesirable degreeof histamine release are typified by cutaneous flushing about the face,neck and/or chest, sometimes accompanied by hypotension and/ortachycardia and/or nausea and vomiting. In some cases, the physicalmanifestations of an undesirably high degree of histamine release caninclude very serious and potentially fatal reactions such asbronchospasm, wheezing, and anaphylactoid reactions and anaphylacticshock. An explanation of the reason(s) that these pharmacological agentscause histamine release in vivo has eluded scientists for years.

[0003] Conventional pharmacological agents which are known to cause orsuspected to be capable of causing histamine release includeintravenously administered hypnotics, analgesics, sedatives, optiates,anesthetics, neuromuscular blocking agents (i.e., “neuromuscularblockers”), contrast agents employed in imaging (i.e., radiographiccontrast media, radio imaging agents and other contrast agents,hereinafter collectively “imaging agents”), hormones for diagnosticprocedures, and certain antibiotics, NSAIDs, anticoagulants, ACEinhibitors and benzodiazepine receptor antagonists. These agents may beadministered intravenously as a bolus or rapid infusion, which can, inaddition to their desired therapeutic, diagnostic or medicinal effect,cause the release of histamine. Histamine release is often the mostprevalent adverse reaction of certain of these pharmacological agents.

[0004] Histamine release could occur through both immunologic andnon-immunologic mechanisms. The more immediate or rapid reactionselicited by these pharmacological agents are believed to occur viarelease of histamine via a non-immunologic mechanism. The latter areoften referred to as anaphylactoid reactions.

[0005] The precise mechanism by which these drugs cause the release ofhistamine is not clear. Mast cells and basophils are possible sources ofthe released histamine, but other in vivo sources may also exist.Mechanistic studies, especially studies conducted in vitro with mastcells are complicated by the tremendous heterogeneity that exists notonly between species, but within a single individual. Given the numberof different sources of histamine, it is possible that differentmechanisms in different cells and tissue may be involved at any onetime.

[0006] Clinically, it is known that slowing the rate of injection ofthese agents from 5 seconds to 30 seconds decreases the incidence ofcardiovascular effects typical of histamine release. Slowing the rate ofadministration is currently the preferred method of avoiding the risksassociated with substantial histamine release. However, slowing the rateof administration is not an acceptable course of action in some clinicalsituations. For example, slowing the rate of administration isunacceptable in emergency medical situations, especially when anesthesiaand intubation prior to emergency surgical procedures must occurrapidly. Furthermore, it is known that slowing administration of certainpharmacological agents can disproportionately decrease the speed ofonset of activity and/or the potency of the drug.

[0007] There remains a need in the art for methods of addressing thehistamine release side effect associated with these pharmacologicalagents.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008]FIG. 1 is a plot of the surface tension (mN/m) versus increasingconcentration of(Z)-2-Chloro-1-{3-{(1R,2S)-6,7-dimethoxy-2-methyl-1-[(3,4,5-trimethoxyphenyl)methyl]-1,2,3,4-tetrahydro-2-isoquinolinio}propyl}-4-{3-[(1S,2R)-6,7-dimethoxy-2-methyl-1-(3,4,5-trimethoxyphenyl)-1,2,3,4-tetrahydro-2-isoquinolinio]propyl}-2-butenedioatedichloride (designated “Compound 1”) (mg/mL).

[0009]FIG. 2 is a graph depicting (-▴-) the proton NMR relaxation rate(T₁ value in secs.) for solutions containing 80 mM(Z)-2-Chloro-1-{3-{(1R,2S)-6,7-dimethoxy-2-methyl-1-[(3,4,5-trimethoxyphenyl)methyl]-1,2,3,4-tetrahydro-2-isoquinolinio}propyl}-4-{3-[(1S,2R)-6,7-dimethoxy-2-methyl-1-(3,4,5-trimethoxyphenyl)-1,2,3,4-tetrahydro-2-isoquinolinio]propyl}-2-butenedioate dichloride (designated“Compound 1”), and various concentrations of d₄-citrate (3.13, 6.25,12.5, 25 and 50 mM) in deuterated saline solution at pD 3, and (--) thepharmaceutically-induced histamine release from rat basophilic leukemiacells as a percent of control value, which is induced by exposure of theRBL cells to a formulation containing 160 mM of the same compound, andvarious concentrations of citric acid (5, 10, 25 and 50 mM) in distilledwater at pH 3.

SUMMARY OF THE INVENTION

[0010] According to a first aspect of the invention there is provided amethod for preparing a pharmaceutical formulation containing a histaminereleaser and a physiologically acceptable excipient. The methodcomprises combining a therapeutically effective amount of the histaminereleaser with a concentration of the physiologically acceptableexcipient. The concentration of the physiologically acceptableexcipient, when combined in an aqueous solution with the histaminereleaser at or above critical micelle concentration, is sufficient toreduce aggregation of the histamine releaser in the aqueous solution byat least about 25 percent compared to aggregation of the histaminereleaser in the aqueous solution containing substantially nophysiologically acceptable excipient.

[0011] According to a second aspect of the invention, there is providedanother method for preparing a pharmaceutical formulation containing ahistamine releaser and a physiologically acceptable excipient. Themethod comprises combining a therapeutically effective amount of thehistamine releaser with the physiologically acceptable excipient. Thephysiologically acceptable excipient is present in a concentrationdetermined by a method comprising the steps of: a) measuring aggregationof the histamine releaser in a reference solution consisting essentiallyof the histamine releaser in a concentration at or above the criticalmicelle concentration in an aqueous solution; b) measuring aggregationof the histamine releaser in a comparative solution consistingessentially of the histamine releaser and a pre-selected concentrationof the physiologically acceptable excipient in the aqueous solution,wherein the concentration of the histamine releaser in the comparativesolution is substantially the same as the concentration of the histaminereleaser in the reference solution; c) optionally repeating step b) oneor more times with a comparative solution having a differentpre-selected concentration of the physiologically acceptable excipientd) identifying a concentration of physiologically acceptable excipientthat is sufficient to reduce aggregation of the histamine releaser inthe comparative solution by at least about 25 percent compared toaggregation of the histamine releaser in the reference solution. Theidentified concentration of step d) is the concentration of thephysiologically acceptable excipient for combining with the histaminereleaser to prepare the pharmaceutical formulation.

[0012] According to a third aspect, there is provided another method ofpreparing a pharmaceutical formulation containing a histamine releaserand a physiologically acceptable excipient. The method comprisescombining a therapeutically effective amount of the histamine releaserwith a concentration of physiologically acceptable excipient. Theconcentration of the physiologically acceptable excipient is determinedby a method comprising the steps of: a) measuring histamine release froma histamine-containing biological sample in a reference mixtureconsisting essentially of: i) the histamine-containing biological samplein a medium and ii) an aqueous solution of the histamine releaser at aconcentration sufficient to cause histamine release from thehistamine-containing biological sample; b) measuring histamine releasefrom the histamine-containing biological sample in a comparative mixtureconsisting essentially of: i) the histamine-containing biological samplein medium and ii) an aqueous solution of the histamine releaser and apre-selected concentration of the physiologically acceptable excipient,wherein the histamine releaser in the comparative mixture is present ina concentration which is substantially the same as the concentration ofhistamine releaser in the reference mixture of step a); c) optionallyrepeating step b) one or more times with a comparative mixture having adifferent pre-selected concentration of the physiologically acceptableexcipient; and d) identifying a concentration of the physiologicallyacceptable excipient sufficient to reduce histamine release from thehistamine-containing biological sample in the comparative mixture by atleast about 10 percent compared to histamine release from thehistamine-containing biological sample in the reference mixture. Theidentified concentration of step d) is the concentration ofphysiologically acceptable excipient for combining with the histaminereleaser to prepare the pharmaceutical formulation.

[0013] According to a fourth aspect of the present invention, there areprovided methods for determining a concentration of a physiologicallyacceptable excipient for combining with the histamine releaser toprepare the pharmaceutical formulation. These methods are as describedabove in connection with methods for preparing the pharmaceuticalformulations of the present invention.

[0014] According to a fifth aspect of the present invention, there isprovided a method for determining a concentration of a physiologicallyacceptable excipient that is sufficient to suppresspharmaceutically-induced histamine release in an animal being treatedwith a histamine releaser. The method comprises: a) measuringaggregation of the histamine releaser in a reference solution consistingessentially of the histamine releaser in a concentration at or above thecritical micelle concentration in an aqueous solution; b) measuringaggregation of the histamine releaser in a comparative solutionconsisting essentially of the histamine releaser and a pre-selectedconcentration of the physiologically acceptable excipient in the aqueoussolution, wherein the concentration of the histamine releaser in thecomparative solution is substantially the same as the concentration ofthe histamine releaser in the reference solution; c) optionallyrepeating step b) one or more times with a comparative solution having adifferent pre-selected concentration of the physiologically acceptableexcipient; d) identifying a concentration of physiologically acceptableexcipient that is sufficient to reduce aggregation of the histaminereleaser in the comparative solution by at least about 25 percentcompared to aggregation of the histamine releaser in the referencesolution. The identified concentration of step d) is the concentrationof the physiologically acceptable excipient that is sufficient tosuppress pharmaceutically-induced histamine release in an animal beingtreated with a histamine releaser. The foregoing method may also beemployed to determine a concentration of a physiologically acceptableexcipient which when combined in an aqueous solution with the histaminereleaser at or above critical micelle concentration, is sufficient toreduce aggregation of the histamine releaser in the aqueous solution byat least about 25 percent compared to aggregation of the histaminereleaser in an aqueous solution containing substantially nophysiologically acceptable excipient.

[0015] According to a sixth aspect of the present invention, there isprovided another method for determining a concentration of aphysiologically acceptable excipient that is sufficient to suppresspharmaceutically-induced histamine release in an animal being treatedwith a histamine releaser. The method comprises: a) measuring histaminerelease from a histamine-containing biological sample in a referencemixture consisting essentially of: i) the histamine-containingbiological sample in a medium and ii) an aqueous solution of thehistamine releaser at a concentration sufficient to cause histaminerelease from the histamine-containing biological sample; b) measuringhistamine release from the histamine-containing biological sample in acomparative mixture consisting essentially of: i) thehistamine-containing biological sample in medium and ii) an aqueoussolution of the histamine releaser and a pre-selected concentration ofthe physiologically acceptable excipient, wherein the histamine releaserin the comparative mixture is present in a concentration which issubstantially the same as the concentration of histamine releaser in thereference mixture of step a); c) optionally repeating step b) one ormore times with a comparative mixture having a different pre-selectedconcentration of the physiologically acceptable excipient; and d)identifying a concentration of the physiologically acceptable excipientsufficient to reduce histamine release from the histamine-containingbiological sample in the comparative mixture by at least about 10percent compared to histamine release from the histamine-containingbiological sample in the reference mixture. The identified concentrationof step d) is the concentration of the physiologically acceptableexcipient that is sufficient to suppress pharmaceutically-inducedhistamine release in an animal being treated with a histamine releaser.This concentration of excipient is advantageously employed for thepreparation of pharmaceutical formulations of the histamine releaser.The foregoing method may also be employed to determine a concentrationof a physiologically acceptable excipient which when combined in asaline solution with the histamine releaser at or above critical micelleconcentration, is sufficient to reduce aggregation of the histaminereleaser in the saline solution by at least about 25 percent compared toaggregation of the histamine releaser in a saline solution containingsubstantially no physiologically acceptable excipient.

[0016] According to another aspect, the present invention providespharmaceutical formulations comprising a histamine releaser and aconcentration of physiologically acceptable excipient. Thepharmaceutical formulations may be prepared according to any of themethods of the present invention. The concentration of physiologicallyacceptable excipient may be determined according to any of the methodsof the present invention. The present invention provides apharmaceutical formulation comprising a histamine releaser and aphysiologically acceptable excipient wherein the concentration of thephysiologically acceptable excipient is sufficient to suppresspharmaceutically-induced histamine release.

[0017] According to another aspect of the invention there is provided amethod for suppressing pharmaceutically-induced histamine release in ananimal being treated with a histamine releaser. The method comprisesadministering to the animal a pharmaceutical formulation according tothe present invention.

[0018] In another aspect, the present invention provides use of apharmaceutical formulation according to the invention for themanufacture of a medicament for suppressing pharmaceutically-inducedhistamine release in an animal being treated with the histaminereleaser.

[0019] According to another aspect of the invention, there is provided amethod for preventing cardiovascular and respiratory effects mediated bypharmaceutically-induced histamine release in an animal being treatedwith a histamine releaser. The method comprises administering to theanimal a pharmaceutical formulation according to the present invention.

[0020] In another aspect, the present invention provides use of apharmaceutical formulation according to the invention for themanufacture of a medicament for preventing cardiovascular andrespiratory effects mediated by pharmaceutically-induced histaminerelease in an animal being treated with the histamine releaser.

[0021] In yet another aspect, the present invention provides a kit forpreparing a pharmaceutical formulation of a histamine releaser. The kitcomprises a) a physiologically acceptable excipient, and b) instructionsfor preparing the pharmaceutical formulation according to the methods ofthe present invention.

[0022] A preferred histamine releaser for use in the pharmaceuticalformulations and methods of treatment of the present invention is(Z)-2-chloro-1-{3-{(1R,2S)-6,7-dimethoxy-2-methyl-1-[(3,4,5-trimethoxyphenyl)methyl]-1,2,3,4-tetrahydro-2-isoquinolinio}propyl}-4-{3-[(1S,2R)-6,7-dimethoxy-2-methyl-1-(3,4,5-trimethoxyphenyl)-1,2,3,4-tetrahydro-2-isoquinolinio]propyl}-2-butenedioatedichloride and pharmaceutically acceptable salts thereof. Thus, inanother aspect, the present invention provides a pharmaceuticalformulation comprising a therapeutically effective amount of(Z)-2-chloro-1-{3-{(1R,2S)-6,7-dimethoxy-2-methyl-1-[(3,4,5-trimethoxyphenyl)methyl]-1,2,3,4-tetrahydro-2-isoquinolinio}propyl}-4-{3-[(1S,2R)-6,7-dimethoxy-2-methyl-1-(3,4,5-trimethoxyphenyl)-1,2,3,4-tetrahydro-2-isoquinolinio]propyl}-2-butenedioatedichloride and pharmaceutically acceptable salts thereof; an excipientselected from the group consisting of glycine in a concentration of fromabout 10 mg/mL to about 30 mg/mL, citric acid in a concentration of fromabout 25 mM to about 75 mM, EDTA in a concentration of from about 0.1%to about 0.5%, calcium chloride in a concentration of from about 25 mMto about 75 mM, and combinations thereof; and a physiologicallyacceptable diluent, wherein said pharmaceutical formulation is suitablefor intravenous administration. The present invention also providesmethods for suppressing pharmaceutically-induced histamine release in ananimal being treated with(Z)-2-chloro-1-{3-{(1R,2S)-6,7-dimethoxy-2-methyl-1-[(3,4,5-trimethoxyphenyl)methyl]-1,2,3,4-tetrahydro-2-isoquinolinio}propyl}-4-{3-[(1S,2R)-6,7-dimethoxy-2-methyl-1-(3,4,5-trimethoxyphenyl)-1,2,3,4-tetrahydro-2-isoquinolinio]propyl}-2-butenedioate dichloride or a pharmaceutically acceptable saltsthereof.

[0023] These and other aspects of the present invention are describedfurther in the Detailed Description of the Invention, which follows andin the claims.

DETAILED DESCRIPTION OF THE INVENTION

[0024] I. Definitions

[0025] “Pharmaceutical agent” as used herein shall refer to agentshaving therapeutic activity (i.e., agents administered to an animal,preferably a human, for the treatment or prevention of a medicalcondition), agents having diagnostic activity (i.e., agents administeredto an animal, preferably a human, for aiding or assisting in thediagnosis of a medical condition), and agents having other medicinalutility (i.e., agents administered to facilitate medical and/or surgicalprocedures) when administered to an animal, preferably a human (e.g.,neuromuscular blockers, anesthetics, analgesics and the like).

[0026] “Histamine releaser” as used herein refers to a pharmaceuticalagent which is selected from the group consisting of anesthetics,opiates, neuromuscular blockers, imaging agents, hormones for diagnosticprocedures, tricyclic glycopeptide antibiotics, cephalosporinantibiotics, penicillin and penicillin derivative antibiotics,intravenously administered non-steroidal anti-inflammatory agents(NSAIDs), anticoagulants, ACE inhibitors, and benzodiazepine receptorantagonists, which when intravenously administered as a rapid bolus orrapid infusion to an animal, causes elevation of plasma and/or tissueconcentrations of histamine above normal physiological levels. Thehistamine releasers are characterized by a structure having one or morehydrophilic charged (cationic or anionic) portions distanced from one ormore hydrophobic portions. More particularly, “histamine releasers”include pharmaceutical agents which when intravenously administered as arapid bolus or rapid infusion to an animal cause a histamine release invivo which is sufficient to produce physiological manifestationsselected from the group consisting of cutaneous flushing, itching,hives, edema, nausea, vomiting, elevated gastric acid secretion,vestibular effects, cardiovascular effects such as hypotension (fall inblood pressure), tachycardia (rise in heart rate), and respiratoryeffects such as bronchoconstriction, anaphylactoid reactions andanaphylactic shock, and combinations of any two or more of theforegoing. “Normal physiological levels of histamine” can differ amongspecies and among individual members of a single species. Therefore,“normal physiological levels of histamine refers to an average plasmahistamine level of an untreated animal of the same species as that beingtreated with the histamine releaser. Normal physiological levels ofhistamine of various species of animals is reported in INFLAMMATION:BASIC PRINCIPLES AND CLINICAL CORRELATES (Eds. J. I. Gallin, I. M.Goldstein, and R. Snyderman, Chap. 11, Measurement of Histamine, p. 202,Raven Press, New York, 1992; and Bertini, S. et al., Gen. Pharmac.31:625-631 (1998).

[0027] As used herein, the term “therapeutically effective amount of ahistamine releaser” means an amount of the pharmaceutical agent which isa histamine releaser (defined above), which amount is sufficient toachieve the desired pharmaceutical activity (i.e., therapeutic activity,diagnostic activity or medicinal utility) of the agent. Thus, in theembodiment wherein the histamine releaser is a neuromuscular blocker, a“therapeutically effective amount of the histamine releaser” is theamount of the neuromuscular blocker which is sufficient to causeskeletal muscle relaxation in the animal to which the neuromuscularblocker is being administered. In the embodiment wherein the histaminereleaser is an anesthetic, a “therapeutically effective amount of thehistamine releaser” is the amount of anesthetic which is sufficient toinduce anesthesia in the animal to which the anesthetic is beingadministered. In the embodiment wherein the histamine releaser is a animaging agent, a “therapeutically effective amount of the histaminereleaser” is the amount of imaging agent which is sufficient to producean appropriate level of image contrast in a diagnostic procedure in theanimal to which the imaging agent is being administered. One skilled inthe art can readily determine the therapeutically effective amount of aparticular histamine releaser based upon the foregoing explanation andexamples, and conventional knowledge in the art regarding thesepharmaceutical agents.

[0028] The term “physiologically acceptable excipient” means an agent,other than water, which is utilized in the formulation of apharmaceutical agent as a pharmaceutical formulation, which is notdeleterious to the animal to which the formulation will be administered,and which does not substantially affect the pharmaceutical activity ofthe pharmaceutical agent with which it is formulated. Typically, thephysiologically acceptable excipients are employed for the purpose offacilitating formulation of the pharmaceutically active agent.

[0029] The term “aqueous solution” as used herein refers to solutionscontaining water (including deuterated water), preferably distilledwater, and “saline solutions” (defined below), and which solutionscontain substantially no other additives, but which may be pH adjustedwith hydrochloric acid or sodium hydroxide, as may be necessary ordesirable to stabilize or facilitate solubilization of certain histaminereleasers in aqueous solution.

[0030] The term “saline solution” as used herein refers to solutionscontaining approximately 0.9% sodium chloride solubilized in water(including deuterated water), preferably distilled water, and whichsolutions contain substantially no other additives but may be pHadjusted with hydrochloric acid or sodium hydroxide as may be necessaryor desirable to stabilize or facilitate solubilization of certainhistamine releasers in the saline solution.

[0031] The term “aggregation” as used herein refers to the averageaggregate size of a pharmaceutical agent, solubilized in aqueoussolution. Aggregate size can be a function of the conformation of themolecules or aggregates, or the number of molecules forming theaggregate, or the hydrodynamic radius of the aggregate.

[0032] II. Histamine Release Generally

[0033] Although the reason(s) that these histamine releasers inducehistamine release have eluded scientists for years, our studies suggestthat histamine release is the result of a combination of at least two,possibly related, factors; namely, the concentration of the histaminereleaser and certain structural properties of histamine releasers thatcan cause aggregation of the histamine releaser in solution and in theblood upon intravenous administration to an animal.

[0034] It is now believed that histamine release may be related to theinitial bolus concentration of the histamine releaser, and that thecritical events leading to histamine release take place very soon afterthe injection. Slowing the rate of injection effectively lowers theconcentration of the histamine releaser since intravenously injectedhistamine releasers are diluted by the flow of blood that passes theinjection site while the agent is being injected.

[0035] By studying surface tension properties of histamine releasers, ithas been found that certain histamine releasers tend to self-associate,or aggregate in aqueous solution. It is now believed that thisaggregation of the histamine releaser triggers the histamine release. Wehave identified structural properties shared by the various histaminereleasers, which may cause aggregation.

[0036] The histamine releasers referred to in the present invention allshare the common structural features of one or more hydrophilic portionsdistanced from one or more hydrophobic portions. For example,neuromuscular blockers are bis-quaternary ammonium salts which possesstwo cationic charges at the ends of the molecule, separated by ahydrophobic, lipophilic linker. In the case of non-steroidal (e.g.,benzylisoquinoline-type) neuromuscular blockers, the hydrophobic linkeris typically long and flexible. In the case of steroidal neuromuscularblockers, the hydrophobic linker may be bulky and/or rigid.

[0037] Because of this structural characteristic, the histaminereleasers are soluble in both water and organic solvents. The structuraland solubility characteristics of histamine releasers are similar tosurfactants and detergents. Surfactants and detergents are known toaggregate in solution in a concentration dependent manner. The presenceof a charged hydrophilic portion distanced from a hydrophobic portion ofthe molecule may impose on the histamine releaser a tendency toself-solvate or aggregate in solution just as a detergent or surfactant,thus explaining the observation of surface tension modifying propertiesof the histamine releasers.

[0038] Our studies indicate that histamine releasers may aggregate insolution. Possible arrangements of aggregates in solution include butare not limited to dimers, trimers, micelles, rods, plates and sheets.In general, histamine releasers tend to aggregate in arrangements whichseek to isolate the hydrophobic portion(s) of the molecule from theaqueous solution in which it is dissolved. This can be accomplished, forexample, by forming micelles wherein the hydrophobic portion of thehistamine releaser molecule is oriented toward the center of the micelleand the charged hydrophilic portion of the molecule is oriented outwardfrom the center of the micelle. For example, the molecules of aneuromuscular blocker could bend in the region of the hydrophobicportion allowing multiple molecules to aggregate by positioning thehydrophobic portion of each molecule in proximity to the hydrophobicportions of other molecules, while extending the cationic, hydrophilicportion outward. This aggregated arrangement of neuromuscular blockermolecules results in aggregation with poly-cationic surfaces extendingoutward from the center of the aggregate. It has been found that thedegree of aggregation is concentration dependent, such that higherconcentrations of histamine releaser result in more aggregation and/orhigher order aggregates (e.g., micelles as compared to dimers).

[0039] Many pharmaceutical agents, including most neuromuscular blockersare administered intravenously at concentrations in the millimolar (mM)range; a concentration range at which it is likely the drug is partlyaggregated and may be near its critical micelle concentration. The“critical micelle concentration” is the concentration at which moleculesin a given environment aggregate to form micelles. The critical micelleconcentration of a given agent can be measured using the techniquesdescribe herein as well as other conventional techniques, includingthose described in Anacker, E. W. (1970), MICELLE FORMATION OF CATIONICSURFACTANTS IN AQUEOUS MEDIA, Cationic Suffactants, E. Jungermann. NewYork, Marcel Dekker, Inc.; Attwood, D. (1995), Advances in Colloid andInterface Science 55: 271-303; and Mukerjee, P. and K. J. Mysels (1971),CRITICAL MICELLE CONCENTRATIONS IN AQUEOUS SURFACTANT SYSTEMS,Washington, D.C., U.S. Dept. of Commerce, the disclosures of which areincorporated herein by reference.

[0040] As is known by those skilled in the art, critical micelleconcentration is dependent upon a number of factors. See, Attwood, D.and A. T. Florence (1983), SURFACTANT SYTEMS: THEIR CHEMISTRY, PHARMACY,AND BIOLOGY, London, Chapman and Hall; Rosen, M. J. (1978), SURFACTANTSAND INTERFACIAL PHENOMENA, New York, Wiley-Interscience; Jungermann, E.,Ed. (1970), CATIONIC SURFACTANTS, New York, Marcel Dekker, Inc.; andJonsson, B., B. Lindman, et al. (1998), SURFACTANTS AND POLYMERS INAQUEOUS SOLUTION, Chichester, John Wilet Et Sons (the disclosures ofwhich are incorporated herein by reference) for a discussion of thefactors which can effect critical micelle concentration. One such factoris the nature of the solution or suspension in which the aggregates areforming. For example, the data obtained for(Z)-2-chloro-1-{3-{(1R,2S)-6,7-dimethoxy-2-methyl-1-[(3,4,5-trimethoxyphenyl)methyl]-1,2,3,4-tetrahydro-2-isoquinolinio}propyl}-4-{3-[(1S,2R)-6,7-dimethoxy-2-methyl-1-(3,4,5-trimethoxyphenyl)-1,2,3,4-tetrahydro-2-isoquinolinio]propyl}-2-butenedioatedichloride, an ultra-short acting neuromuscular blocker in water isconsistent with a critical micelle concentration at about 15 mg/mL(about 14 mM), while in saline the data obtained for this drug isconsistent with a critical micelle concentration at between 40 and 80mM. The concentration of neuromuscular blockers used clinicallytypically ranges from about 1 to about 55 mM.

[0041] Aggregation of the histamine releaser and the critical micelleconcentration are highly dependent on many factors. One factor is thestructure of the particular histamine releaser molecule, including thepresence and structure of one or more hydrophobic domains distanced fromone or more hydrophilic domains that often contain cationic or anionicgroups. In addition, aggregation and critical micelle concentration arehighly dependent on the concentration of the histamine releaser insolution, the presence and concentration of other molecules in solution,pH, the identity and valency of the counter-ion to any cationic oranionic groups, and on the temperature and pressure of the solution.

[0042] Upon injection of the histamine releasers into blood, theresultant new solution (i.e., the solution of histamine releaser inblood) is believed to change many of the factors listed above veryrapidly. Given the rapidly changing solution conditions such as theionic composition of blood and the presence of many other dissolvedsolutes, cells, proteins, etc., it is reasonable to expect theseconditions would favor increased aggregation and/or a lower criticalmicelle concentration. Overall, it is likely that these histaminereleasers are already highly aggregated in the formulation beingadministered and that the physical act of mixing with blood in the veinupon injection may favor increased aggregation and/or the formation ofmicelles.

[0043] Aggregated histamine releaser molecules are now believed toinduce histamine release from cells, tissues and fluids in essentiallythe same manner as aggregated molecules of surfactants. Aggregatedmolecules of surfactants can cause a detergent action that cansolubilize organic molecules in water, extract proteins from membranes,and cause cell membranes to become permeable. Increasing thepermeability of the membrane makes it easier for molecules to enter thecell, and for molecules in the cell, such as histamine molecules, to bereleased.

[0044] It has now been observed that histamine releasers tend toaggregate. These aggregates may exhibit detergent-like properties.Accordingly, it is believed that the detergent-like properties of theaggregated histamine releaser may cause cell membranes to becomedisturbed possibly to the point that the cells rupture. The disturbanceof cellular membranes of histamine containing cells, or in tissues orfluids and/or rupture of these cells may cause the release ofintracellular components and molecules into the surrounding environment.Surfactants and detergents are known to cause the release of histamineby rupturing the cell membrane of mast cells, which are known to storeand release histamine. Other cell types may behave similarly. Forexample, basophils present in blood are also known to store and releasehistamine and would be expected to behave similar to mast cells whenexposed to a surfactant or aggregated histamine releaser.

[0045] Even lower order aggregates (i.e., dimers and trimers) may causehistamine release that may or may not be related to detergent-likeeffects. See, Read, G. W. and J. F. Lenney, Journal of MedicinalChemistry 15(3): p. 320-23 (1972). For example, because bis-cationicagents such as neuromuscular blockers, carry two ammonium groups permolecule, it is currently expected that histamine releasing effects canbe expected when as few as 2-4 molecules of neuromuscular blockeraggregate.

[0046] In addition to this direct mechanism explaining the release ofhistamine, it is also possible that histamine release can be caused byindirect mechanisms. Cells that do not store histamine, such asendothelial cells lining blood vessels, may cause the release ofhistamine by an indirect mechanism. Aggregates of histamine releasersmay cause the release of cellular components from endothelial cells (orother cells which do not store histamine), and those components maytravel to histamine-containing cells and signal the release of histaminefrom those storage cells.

[0047] III. Methods of Making Formulations

[0048] The present invention provides several methods of makingpharmaceutical formulations containing a histamine releaser and aphysiologically acceptable excipient, which seek to address the abovementioned tendencies of histamine releasers to aggregate and cause therelease of undesirable levels of histamine upon administration in vivo.

[0049] A. Histamine Releasers

[0050] In particular, the histamine releasers referred to in the presentinvention and which are employed in the methods of making pharmaceuticalformulations, include anesthetics, opiates, neuromuscular blockers,imaging agents, hormones for diagnostic procedures, tricyclicglycopeptide antibiotics, cephalosporin antibiotics, penicillin andpenicillin derivative antibiotics, intravenously administerednon-steroidal anti-inflammatory agents (NSAIDs), anticoagulants, ACEinhibitors, and benzodiazepine receptor antagonists, which have thestructural features noted above in the definition of the “histaminereleaser.” Preferably, the histamine releaser is selected from the groupconsisting of anesthetics, opiates, neuromuscular blockers, and imagingagents. More preferably the histamine releaser is a neuromuscularblocker.

[0051] Examples of anesthetics include but are not limited to thiopentaland thiobutabarbital. Examples of opiates include but are not limited tomorphine, morpholinic derivatives such as oxymorphone, nalbuphinehydrochloride, buprenorphine, hydromorphone, fentanil and fentanilderivatives including remifentanil, sufentanil and alfentanil. Examplesof neuromuscular blockers include but are not limited to non-steroidalneuromuscular blocking agents such as(Z)-2-chloro-1-{3-{(1R,2S)-6,7-dimethoxy-2-methyl-1-[(3,4,5-trimethoxyphenyl)methyl]-1,2,3,4-tetrahydro-2-isoquinolinio}propyl}-4-{3-[(1S,2R)-6,7-dimethoxy-2-methyl-1-(3,4,5-trimethoxyphenyl)-1,2,3,4-tetrahydro-2-isoquinolinio]propyl}-2-butenedioatedichloride (hereinafter sometimes referred to as “Compound 1” for thesake of brevity), mivacurium, atracurium, d-tubocurarine, metocurine,doxacurium, and gallamine; steroidal neuromuscular blocking agents suchas vecuronium, pancuronium, rocuronium, and rapacuronium; and otherneuromuscular blockers such as succinylcholine. Examples of imagingagents include but are not limited to ioxaglic (ioxaglate) acid anddiatrizoate sodium.

[0052] Pharmaceutically acceptable salts of any of the foregoing arealso contemplated by the present invention. Thus, the present inventioncontemplates the use of atracurium besylate, mivacurium chloride,vecuronium bromide, pancuronium bromide, rapacuronium bromide,doxacurium chloride, succinylcholine chloride, morphine sulphate,hydromorphone hydrochloride, pharmaceutically acceptable salts ofCompound 1, and the like.

[0053] Combinations of any two or more of the above-referenced histaminereleasers into a single formulation are also contemplated, provided thatthe two or more histamine releasers do not react in a manner whichdeleteriously impacts their therapeutic activity.

[0054] Preferred histamine releasers for use in the present inventioninclude morphine, Compound 1, mivacurium, atracurium, vecuronium,pancuronium, rocuronium, rapacuronium and succinylcholine chloride andpharmaceutically acceptable salts thereof. Compound 1 and pharmaceuticalformulations and methods of treatment using this compound are describedin PCT Publication Nos. 98/42674 and 98/42675, both published Oct. 1,1998, to Glaxo Wellcome Inc. and Cornell Research Foundation, thesubject matter of which are hereby incorporated by reference in theirentirety.

[0055] In one preferred embodiment, the histamine releaser is Compound 1or a pharmaceutically acceptable salt thereof. In one preferredembodiment, the histamine releaser is mivacurium or a pharmaceuticallyacceptable salt thereof. In one preferred embodiment, the histaminereleaser is atracurium or a pharmaceutically acceptable salt thereof,e.g., atracurium besylate.

[0056] B. Physiologically Acceptable Excipients

[0057] According to the methods of the present invention the histaminereleaser is combined with a physiologically acceptable excipient, whichmay be a single physiologically acceptable excipient or a combination oftwo, three or more physiologically acceptable excipients. “Excipient” asused herein means both a single physiologically acceptable excipient anda combination of two, three or more physiologically acceptableexcipients.

[0058] Preferred excipients are those which are conventionally employedin approved parenteral or injectable formulations. The excipientsemployed in the present invention are physically characterized by thepresence of charge (e.g., ionic excipients) and/or the presence oforganic residues which are capable of affecting the solvation of thehistamine releaser. More particularly, the excipients are capable ofionizing in solution and/or may be solvating the histamine releaser oraiding in the salvation of the histamine releaser.

[0059] Suitable excipients for use in the present invention may beselected from a variety of categories, including but not limited todivalent inorganic salts (i.e., inorganic salts having a divalent anion,a divalent cation, or both), organic carboxylic acids, phosphoric acid,amino acids, chelating agents, albumins and combinations thereof.Preferred excipients are selected from the group consisting of divalentinorganic salts, organic carboxylic acids, phosphoric acid, amino acids,chelating agents, albumins and combinations thereof. In one embodiment,the excipient is a divalent inorganic salt. In one embodiment, theexcipient is an organic acid. In one embodiment, the excipient is achelating agent. In one embodiment, the excipient is an amino acid.

[0060] Examples of suitable divalent inorganic salts include but are notlimited to calcium chloride, magnesium sulphate, magnesium chloride,sodium sulphate, and combinations thereof. Calcium chloride is apreferred excipient for use in the present invention.

[0061] Examples of suitable organic carboxylic acids include but are notlimited to tartaric acid (which includes racemic tartaric acid,D-tartaric acid and L-tartaric acid) maleic acid, acetic acid, citricacid, succinic acid, glucuronic acid, and combinations thereof. “Citricacid” as used herein refers to citric acid and any hydrates and saltsthereof, i.e., citrates. Citric acid is a preferred excipient for use inthe present invention.

[0062] The term “phosphoric acid” as used herein also includes salts ofphosphoric acid, such as sodium phosphate. As will be readily apparentto one skilled in the art, the use of phosphoric acid as an excipient inthe instant invention will require that it be provided in aconcentration which is physiologically acceptable.

[0063] Examples of suitable amino acids include but are not limited toglycine, lysine, arginine and combinations thereof. Glycine is apreferred excipient for use in the present invention.

[0064] Examples of suitable chelating agents include but are not limitedto ethylene diamine tetracetic acid (EDTA), salts of EDTA (includingEDTA-disodium and EDTA-calcium disodium) and combinations thereof.“EDTA” as used herein, shall refer to EDTA and any salts thereof. EDTAis a preferred excipient for use in the present invention.

[0065] Examples of suitable albumins include bovine serum albumin, humanserum albumin and combinations thereof.

[0066] One skilled in the art will appreciate that additional excipientsmay be employed, which exhibit the physical characteristics noted aboveand which are capable of reducing aggregation of the histamine releaserin aqueous solution and/or otherwise suppressingpharmaceutically-induced histamine release. Such other excipients may beascertained by utilizing the techniques described below regarding thedetermination of appropriate excipient concentrations for use in thepresent invention, and are therefore contemplated by the presentinvention.

[0067] The excipient may be a combination of any two, three, or moreexcipients all selected from one of the foregoing categories, or acombination of any two, three or more excipients selected from two ormore different categories above. For example, the excipient may be acombination of a divalent inorganic salt and an organic carboxylic acid.In another embodiment, the excipient may be an organic carboxylic acidand a chelating agent. In another embodiment, the excipient may be acombination of a divalent inorganic salt and a chelating agent. In yetanother embodiment, the excipient may be a combination of a divalentinorganic salt, an organic carboxylic acid and a chelating agent. In yetanother embodiment, the excipient may be a combination of a divalentinorganic salt and an amino acid.

[0068] The only requirement for utilizing combinations of excipients inthe methods and formulations of the present invention is that theselected excipients will not interact with each other in a manner whichis deleterious to their ability to function in the methods andformulations of the present invention. Other combinations of two or morephysiologically acceptable excipients will be readily apparent to thoseskilled in the art based upon the foregoing examples, and arecontemplated by the instant invention. The techniques described belowfor the determination of appropriate excipient concentrations areequally applicable for the determination of various other combinationsof two, three or more excipients which can be employed in the presentinvention.

[0069] In one preferred embodiment, the excipient is a combination ofany two or more (i.e., two, three or all four) excipients selected fromthe group consisting of glycine, EDTA, citric acid and calcium chloride.In one preferred embodiment, the excipient is a combination of citricacid and EDTA. In one preferred embodiment, the excipient is acombination of citric acid and calcium chloride. In one preferredembodiment, the excipient is a combination of glycine and EDTA. In onepreferred embodiment, the excipient is a combination of glycine andcitric acid. In one preferred embodiment, the excipient is a combinationof citric acid, glycine and calcium chloride.

[0070] C. Methods for Determining a Suitable Concentration of Excipient

[0071] According to the methods of the present invention, pharmaceuticalformulations are prepared by combining a therapeutically effectiveamount of the histamine releaser with a concentration of thephysiologically acceptable excipient. Preferred excipients for use inthese methods of the present invention may be selected from the groupconsisting of divalent inorganic salts, organic carboxylic acids,phosphoric acid, amino acids, chelating agents, albumins andcombinations thereof. In general, the concentration of the excipient isa concentration which is sufficient to suppress pharmaceutically-inducedhistamine release in an animal being treated with a histamine releaser.In one embodiment, the concentration of the excipient is a concentrationwhich when combined in an aqueous solution with the histamine releaserat or above the critical micelle concentration of the histaminereleaser, is sufficient to reduce aggregation of the histamine releaserin the aqueous solution by at least about 25 percent compared toaggregation of the histamine releaser at the same concentration in theaqueous solution containing substantially no excipient.

[0072] “Substantially no excipient” means that the solution or mixtureis substantially free of excipient, that is, it does not contain anamount of excipient that is sufficient to reduce aggregation of thehistamine releaser in solution or suppress pharmaceutically-inducedhistamine release in vitro or in vivo. Preferably, “substantially noexcipient” means that the solution or mixture does not contain an amountof excipient that is sufficient to either reduce aggregation of thehistamine releaser by more than 10 percent or suppresspharmaceutically-induced histamine release in vitro or in vivo, by morethan 5 percent.

[0073] In other words, in an aqueous solution containing the histaminereleaser at or above its critical micelle concentration and one or morephysiologically acceptable excipients in suitable concentrations,aggregation of the histamine releaser will be at least about 25 percentlower than aggregation of the histamine releaser in an aqueous solutioncontaining substantially the same concentration of histamine releaserand substantially no physiologically acceptable excipient. The inclusionof the physiologically acceptable excipient in the solution containingthe histamine releaser substantially reduces aggregation of thehistamine releaser in the solution.

[0074] “Reduce(d) aggregation,” “lower(ed) aggregation” or “decrease(d)aggregation” refers to a decrease in average aggregate size (which canbe a function of conformation of the molecules or aggregates or thenumber of molecules forming the aggregate) and/or a decrease in thenumber of aggregates per unit volume. Methods for measuring aggregationof the histamine releaser and thereby identifying a concentration ofexcipient which is suitable for preparing the formulations of thepresent invention, are discussed below. Preferably, the concentration ofthe excipient will be a concentration which is sufficient, for theparticular histamine releaser and excipient employed, to reduceaggregation of the histamine releaser in the aqueous solution by atleast about 50 percent, and more preferably by at least about 60percent, as compared to an aqueous solution of histamine releasercontaining substantially no excipients.

[0075] In another embodiment, the concentration of the excipient is aconcentration which is sufficient to substantially reduce histaminerelease from a histamine-containing biological sample in a mixture ofthe histamine-containing biological sample in medium and an aqueoussolution containing both the histamine releaser in a concentrationsufficient to cause histamine releaser from the biological sample andthe physiologically acceptable excipient, compared to histamine releasefrom the histamine-containing biological sample in a mixture consistingof the histamine-containing biological sample in medium, and an aqueoussolution containing substantially the same concentration of thehistamine releaser, and substantially no excipient. That is, histaminerelease from the biological sample is reduced by at least about 10percent compared to the reference mixture which contains substantiallyno excipient.

[0076] In other words, a mixture of the histamine containing-biologicalsample in medium and an aqueous solution containing the histaminereleaser at a concentration sufficient to cause histamine release fromthe histamine-containing biological sample and the physiologicallyacceptable excipient in a suitable concentration, will exhibit histaminerelease from the histamine-containing biological sample, which is atleast about 10 percent lower than histamine release from ahistamine-containing biological sample in a mixture of thehistamine-containing biological sample in medium and an aqueous solutioncontaining the same concentration of histamine releaser andsubstantially no physiologically acceptable excipient. Methods formeasuring histamine release from the histamine-containing biologicalsample and methods for identifying a concentration of excipient which issuitable for preparing the formulations of the present invention basedupon the measured histamine release from the histamine-containingbiological sample, are discussed further below. Preferably, theconcentration of the excipient will be a concentration which issufficient, for the particular histamine releaser and excipientemployed, to reduce histamine release from the histamine-containingbiological sample by at least about 20 percent and more preferably by atleast about 25 percent, as compared to histamine release from thehistamine-containing biological sample in a mixture consisting of thehistamine-containing biological sample in medium, and an aqueoussolution containing the histamine releaser at substantially the sameconcentration and substantially no excipient.

[0077] In yet another embodiment, the concentration of the excipient isa concentration which, when intravenously administered together with ahistamine releaser to an animal, is sufficient to suppress one or moreof the physical manifestations of plasma and/or tissue concentrations ofhistamine that are above normal physiological levels of histamine. Thesuppression of one or more physical manifestations of plasma and/ortissue concentrations of histamine above normal physiological levels canbe observed qualitatively or quantitatively, and is discussed furtherbelow.

[0078] The present invention provides multiple methods for determining aconcentration of excipient which is suitable for combining with thehistamine releaser to prepare the pharmaceutical formulations of thepresent invention. A suitable concentration of excipient employed in thepresent invention will depend upon various factors including forexample, the particular histamine releaser with which the excipient willbe combined, the animal which is being treated with the histaminereleaser or to which the pharmaceutical formulation will beadministered, and the particular excipient (or combination ofexcipients) employed. In some cases, pH may play a role in theoptimization of the concentration of excipients as well. In general, thesuitable concentration of the excipient will be a physiologicallyacceptable concentration.

[0079] 1.) Solution-Based Methods

[0080] According to one method of the present invention, theconcentration of excipient is determined by the method comprising thesteps of: a) measuring aggregation of the histamine releaser in areference solution consisting essentially of the histamine releaser in aconcentration at or above the critical micelle concentration in theaqueous solution; b) measuring aggregation of the histamine releaser ina comparative solution consisting essentially of the histamine releaserand a pre-selected concentration of the physiologically acceptableexcipient in the aqueous solution, wherein the concentration of thehistamine releaser in the comparative solution is substantially the sameas the concentration of the histamine releaser in the referencesolution; c) optionally repeating step b) one or more times with acomparative solution having a different pre-selected concentration ofthe physiologically acceptable excipient; d) identifying a concentrationof physiologically acceptable excipient that is sufficient to reduceaggregation of the histamine releaser in the comparative solution by atleast about 25 percent compared to aggregation of the histamine releaserin the reference solution. The identified concentration of step d) isthe concentration of the physiologically acceptable excipient forcombining with the histamine releaser to prepare the pharmaceuticalformulation.

[0081] The order of steps a), b) and c) is not critical. These steps maybe carried out in any desired order, as will be appreciated by thoseskilled in the art. It is not necessary that the aggregation of thehistamine releaser in the reference solution be measured beforeaggregation of the histamine releaser in the comparative solution, andthe present invention contemplates methods comprising these steps in anysuitable order.

[0082] Step c) of optionally, repeating step b) one or more times meansthat step b) may be repeated with multiple comparative solutions, but isnot required. One skilled in the art is capable of determining whetherit is desirable in a particular case to carry out step c), (i.e., repeatstep b)) or not. In general, the preferred methods of the inventioninclude repeating step b) at least once, and more preferably more thanonce.

[0083] The reference solution and comparative solution(s) can beprepared using conventional techniques. For example, the referencesolution can be prepared by solubilizing the histamine releaser, at orabove the critical micelle concentration of the histamine releaser, inaqueous solution.

[0084] One or more comparative solution(s), each including a differentpre-selected concentration of excipient, can be prepared according toseveral different methods. Each comparative solution, after addition ofthe pre-selected concentration of excipient, will have a histaminereleaser concentration which is substantially the same as theconcentration of the histamine releaser in the reference solution.“Substantially the same” concentration means that the difference in theconcentration of the histamine releaser between the reference solutionand the comparative solution is not sufficient to produce a measurableeffect on aggregation of the histamine releaser. More preferably, theconcentration of the histamine releaser in the comparative solution(s)will be considered to be substantially the same as the concentration inthe reference solution when the concentration of the comparativesolution(s) is the concentration of the reference solution plus or minusabout 5%. For the most informative comparison of the aggregation of thehistamine releaser in the reference solution versus aggregation of thehistamine releaser in the comparative solution, each of the referencesolution and the comparative solution(s) will contain an equivalentconcentration of histamine releaser.

[0085] According to one embodiment, comparative solution(s) are preparedby titrating pre-selected, or otherwise known, concentrations of theexcipient into an aqueous solution containing the appropriateconcentration of the histamine releaser. The excipient may be titratedinto the solution containing the histamine releaser by titrating eitherup or down, according to techniques known to those skilled in the art.Thus, the present invention provides a method for determining a suitableconcentration of excipient for combining with the histamine releaser toprepare a pharmaceutical formulation that comprises the steps of: 1)measuring aggregation of the histamine releaser in a reference solutionconsisting essentially of the histamine releaser at or above criticalmicelle concentration in the aqueous solution; 2) titrating thephysiologically acceptable excipient into the reference solution, toprepare comparative solutions; and 3) identifying a concentration ofphysiologically acceptable excipient sufficient to reduce aggregation ofthe histamine releaser in the comparative solution by at least about 25percent compared to the measured aggregation of step 1). The identifiedconcentration is the concentration of the physiologically acceptableexcipient for combining with the histamine releaser to prepare thepharmaceutical formulation. The step of “titrating the excipient” refersto incrementally adding known quantities of excipient (or diluting downto known quantities of excipient) while monitoring the aggregation ofthe histamine releaser in the solution.

[0086] According to another embodiment, comparative solution(s) areprepared by solubilizing a pre-selected concentration of excipient inaqueous solution to create a stock solution having the highestconcentration of excipient, diluting a portion of the stock solution bya suitable dilution factor (e.g., serially diluting the stock solution)one or more times to prepare multiple aqueous solutions of excipienteach containing a pre-selected concentration of excipient, and thensolubilizing the appropriate amount of histamine releaser in eachprepared aqueous solution of excipient to obtain one or more comparativesolutions each having a histamine releaser concentration substantiallythe same as the concentration of histamine releaser in the referencesolution.

[0087] Comparative solutions containing differing concentrations of acombination of two or more excipients may be utilized in the methods ofthe present invention just as comparative solutions containing differingconcentrations of only one excipient. It is well within the purview ofone skilled in the art to prepare multiple comparative solutions, eachcontaining a different pre-selected concentration of two or moredifferent excipients. For example, this can be accomplished by preparingmultiple different comparative solutions each having a differentconcentration of only one of the two or more excipients while theconcentration of the other(s) excipients remain constant. Alternatively,multiple different comparative solutions can be prepared, each having adifferent concentration of more than one, or even all of the excipients.When the excipient employed in the methods and formulations of thepresent invention is a combination of two or more excipients, all thatis required is that the concentration of each of the two or moreexcipients in each comparative solution should be known.

[0088] Steps a) and b) of measuring aggregation of the histaminereleaser in the reference solution and the comparative solution(s), canbe carried out in many different ways. Advantageously, aggregation canbe measured using conventional equipment and techniques known to thoseskilled in the art.

[0089] (a) Surface Tension Analysis

[0090] According to one technique, steps a) and b) are carried out bymeasuring aggregation of the histamine releaser in aqueous solutionusing surface tension analysis. Methods of measuring aggregation bysurface tension are generally described in Anacker, E. W. (1970),MICELLE FORMATION OF CATIONIC SURFACTANTS IN AQUEOUS MEDIA, CATIONICSURFACTANTS, E. Jungermann. New York, Marcel Dekker, Inc.; Iwunze, M.O., M. Lambert, et al. (1997), Monatshefte fur Chemie 128: 582-592;Attwood, D. and R. Natarajan (1979), J. Pharm. Pharmacology 32: 460-462;Moroi, Y. and et.al. (1990), Journal of Physical Chemistry 94: 842-845;and Rosen, M. J., J. H. Mathias, et al. (1999), Langmuir 15(21):7340-7346, the disclosures of which are incorporated herein by referencein their entirety.

[0091] When measuring aggregation by surface tension analysis, theaqueous solution is preferably water (including deuterated water) whichmay be pH adjusted with hydrochloric acid or sodium hydroxide as may benecessary or desirable to physically and/or chemically stabilize orfacilitate the solubilization of certain histamine releasers in theaqueous solution sufficiently long to permit the measurement ofaggregation of the histamine releaser using surface tension analysis.Because the surface tension of most liquids decreases with an increasein temperature, it is necessary to control the temperature of the systemwhile evaluating the surface tension of the reference solution and thecomparative solution(s).

[0092] Aggregation of the histamine releaser in the reference solutionand the comparative solution(s) can be measured by measuring the surfacetension of each of the reference solution and the comparativesolution(s) using the DuNouy ring method, as described in PHYSICALPHARMACY: PHYSICAL CHEMICAL PRINCIPLES IN THE PHARMACEUTICAL SCIENCES,Editors: Alfred Martin, James Swarbrick, Arthur Cammarata, ThirdEdition, Lea Et Febiger, Philadelphia, 1983. Surface tension may providea measure of the critical micelle concentration of the histaminereleaser in solution and for this reason is an indicator of aggregationof the histamine releaser in the solution. The critical micelleconcentration of the histamine releaser in a solution can be identifiedby measuring the surface tension of solutions containing increasingconcentrations of histamine releaser and observing on a plot of surfacetension (y-axis) versus concentration of histamine releaser (x-axis),the point at which an increased concentration of histamine releaserproduces substantially the same or even lower surface tension than thesurface tension of the preceding lower concentration of histaminereleaser. FIG. 1 is a plot of the surface tension of solutionscontaining the histamine releaser, Compound 1, versus increasingconcentration of the Compound 1. The data is consistent with a criticalmicelle concentration at approximately 15 mg/mL of Compound 1.

[0093] It is known that surface tension of a solution containing acompound or material which aggregates will generally decrease as theconcentration of the compound or material in solution increases due toadsorption of the compound or material in solution at the surface of thesolution. Thus, as increasing concentrations of histamine releaser areadded to an aqueous solution, the surface tension of the solution willtend to decrease because of increasing adsorption of the histaminereleaser at the surface of the solution up to the point where thesurface is saturated. Further addition of the histamine releaser to thesolution will result in the formation of aggregates in the bulksolution. It is over a relatively narrow concentration range where thisaggregation or micelle formation occurs and this is the critical micelleconcentration.

[0094] A plot of the surface tension of comparative solutions containinga constant concentration of histamine releaser and various, typicallyincreasing, concentrations of the excipient will enable observation ofan end to or a reversal of the surface tension lowering trend or a shiftin the critical micelle concentration of the histamine releaser. An endto the surface tension lowering trend can be graphically observed by achange in slope in the plot of surface tension v. concentration. Areversal of the surface tension lowering trend can be graphicallyobserved by a change in direction of the plot of surface tension v.concentration. The end in or reversal of the surface tension loweringtrend indicates a reduction in the adsorption of the histamine releaserto the surface of the solution in the comparative solution as comparedto the adsorption of the histamine releaser to the surface of thesolution in the reference solution. This reduction in turn correlates toa reduction of aggregation of the histamine releaser in solution.

[0095] A shift in the critical micelle concentration of the histaminereleaser in the comparative solution(s) is another means of identifyinga reduction in the aggregation of the histamine releaser by surfacetension analysis. A shift in the critical micelle concentration of thehistamine releaser in the comparative solution is identified by theobservation of a higher critical micelle concentration for the histaminereleaser in the comparative solution as compared to the critical micelleconcentration of the histamine releaser in the reference solution. Thus,a reduction in aggregation can be identified by observing a criticalmicelle concentration of the histamine releaser in the comparativesolution occurring at a higher concentration of histamine releaser thanthe critical micelle concentration of the histamine releaser in thereference solution which contains substantially no physiologicallyacceptable excipient.

[0096] For purposes of comparing aggregation of the histamine releaserin the reference solution to aggregation of the histamine releaser inthe comparative solution(s), the surface tension properties of thesolutions can be observed as a plot or curve of surface tension (y-axis)versus concentration of excipient (x-axis). The reference solution andthe comparative solution(s) each represent one point on the surfacetension versus concentration curve. In this manner, trends in thesurface tension of the solutions as the concentration of excipient isaltered (i.e., raised or lowered) can be readily observed.

[0097] (b) NMR Methods

[0098] According to another technique, steps a) and b) are carried outby measuring aggregation of the histamine releaser in aqueous solutionusing nuclear magnetic resonance spectroscopy (NMR). Methods ofmeasuring aggregation by NMR are described in Wiedmer, S. K., M. L.Riekkola, et al. (1997), Analytical Chemistry 69(8): 1577-1584 andJonsson, B., B. Lindman, et al. (1998), SURFACTANTS AND POLYMERS INAQUEOUS SOLUTION. Chichester, John Wilet

Sons, the disclosures of which are incorporated herein by reference intheir entirety.

[0099] Proton NMR spectra of the histamine releaser in aqueous solutioncan be measured using conventional techniques known to those skilled inthe art of NMR spectroscopy on a conventional NMR spectrometer. Basedupon the spectra obtained, chemical shift changes (See, Y. S. Lee, etal., Bull. Korean Chem. Soc., 1993, 14(3), 392-398) line width changes(See, J. H. Bradbury, et al., Nature, 1968, 218, 1049-1050; L. Hwang, etal., J. Phys. Chem., 1988, 92, 4753-4758), relaxation (T₁ and T₂relaxation rate constants) (See, Y. S. Lee, et al., Bull. Korean Chem.Soc., 1993, 14(3), 392-398; B. P. Hills, et al., Macromolecules, 1991,24, 2944-2950), and diffusion coefficients (See, K. F. Morris, et al.,JACS, 1993, 115, 4291-4299; P. Stilbs, Prog. Nucl. Magn. Reson.Spectrosc., 1987, 19,1-45) can be measured. Typically, aggregation ofthe histamine releaser in the reference solution and the comparativesolution is measured by NMR relaxation.

[0100] There are two types of NMR relaxation, spin-lattice and spin-spinrelaxation, referred to as T₁ and T₂ values, respectively. T₁ and T₂ arerate constants which measure the relaxation of NMR magnetization back toequilibrium (hereinafter collectively referred to as “NMR relaxationrates”). For small molecules in what is called the extreme narrowinglimit, both T₁ and T₂ increase in inverse proportion to the rotationaltumbling rate t_(c), which, in turn, is directly proportional to theaverage size of the aggregate. Thus, for small molecules, increases inT₁ and/or T₂ rates with the addition of excipient to the solutioncontaining the histamine releaser, in the absence of significant changesin the viscosity of the solution, indicates a decrease in aggregation.

[0101] According to one embodiment, the steps a) and b) of measuringaggregation of the histamine releaser in the reference solution andcomparative solution(s) comprise measuring proton NMR relaxation ratesof the histamine releaser in the reference solution and the comparativesolution.

[0102] The aggregation of the histamine releaser in each comparativesolution is measured and compared to the measure of aggregation of thehistamine releaser in the reference solution. Comparison to the measureof aggregation of the histamine releaser in the reference solution willenable the ready identification of the comparative solution(s), whichexhibit a measure of aggregation of the histamine releaser which is atleast about 25 percent lower than the measure of aggregation of thehistamine releaser in the reference solution. The step of identifyingthe concentration of physiologically acceptable excipient which issufficient to reduce aggregation of the histamine releaser in thecomparative solution(s) by at least about 25 percent compared toaggregation of the histamine releaser in the reference solution may beachieved after comparing the measured aggregation of the histaminereleaser in each of the reference solution and the comparativesolution(s), as the concentration of excipient in each comparativesolution is pre-selected or known. Thus, in one embodiment of the methodfor determining the concentration of excipient, step d) of identifying aconcentration of physiologically acceptable excipient that is sufficientto reduce aggregation of the histamine releaser in the comparativesolution by at least about 25 percent compared to the measuredaggregation of the histamine releaser in the reference solution,comprises identifying a concentration of physiologically acceptableexcipient sufficient to slow the measured NMR relaxation rates of thehistamine releaser in the comparative solution by at least about 25percent, more preferably about 50 percent and most preferably about 60percent, compared to the measured NMR relaxation rates of the histaminereleaser in the reference solution. A longer time in seconds for themeasured T₁ and T₂ values, is indicative of a slower relaxation rate. Aswill be apparent to those skilled in the art, viscosity changes shouldbe taken into account when measuring aggregation with relaxation rates.

[0103] Carbon-13 nuclear magnetic resonance may also be used in steps a)and b) to measure aggregation of the reference solution and comparativesolution(s) in order to determine concentrations of excipient that aresuitable for preparation of the pharmaceutical formulations of theinvention. This method involves measuring the relaxation rates orchemical shifts changes for carbon rather than proton and has anadvantage in that carbon relaxation is typically less complicated thanproton relaxation and thus is often easier to interpret. However, it isnoted that ¹³C-NMR is 100 times less sensitive than ¹H-NMR. In addition,the measurements can be made for other NMR-active nuclei (i.e. ³¹P, ¹⁹F)if the structure of the histamine releaser contains these nuclei. Theforegoing methods of preparing solutions, measuring aggregation andidentifying the concentration of excipient suitable for use in thepharmaceutical formulations of the invention are equally applicable to¹³C-NMR and for other NMR-active nuclei.

[0104] As noted above, diffusion coefficients can also be measured byNMR and can provide another measure of aggregation in addition to orinstead of the relaxation rate measure discussed above. Diffusioncoefficients reflect changes in average aggregate size and/or the numberof aggregates and therefore are a meaningful measure of aggregation ofthe histamine releaser in the reference solution and the comparativesolution(s). As will be apparent to those skilled in the art, viscositychanges should be taken into account when measuring aggregation withdiffusion coefficients.

[0105] The foregoing methods for measuring aggregation of the histaminereleaser in the reference solution and the comparative solution(s) andidentifying a concentration of excipient which is sufficient to reduceaggregation by at least about 25 percent are illustrative of the methodsof the present invention, but are not exhaustive of all possible ways ofperforming this method of the present invention. One skilled in the artwill readily appreciate the many ways that aggregation of the histaminereleaser may be measured, as the foregoing examples demonstrate that anysuitable technique for measuring aggregation of a molecule in solutionwill be useful in the methods of the present invention for measuringaggregation of a histamine releaser as well. Additional methods (andreferences describing such methods) for measuring aggregation includethe following, which are incorporated herein by reference:

[0106] interfacial tension (see, Rosen, M. J., J. H. Mathias, et al.(1999), Langmuir 15(21): 7340-7346);

[0107] light scattering (see, Anacker, E. W. (1970), MICELLE FORMATIONOF CATIONIC SURFACTANTS IN AQUEOUS MEDIA. CATIONIC SURFACTANTS, E.Jungermann, New York, Marcel Dekker, Inc.; Attwood, D. and R. Natarajan(1979), J. Pharm. Pharmacology 32: 460-462; Moroi, Y. et.al. (1990),Journal of Physical Chemistry 94: 842-845; Moroi, Y., Y. Murata, et al.(1992), Journal of Physical Chemistry 96: 8610-13; and Rosen, M. J.(1978), SURFACTANTS AND INTERFACIAL PHENOMENA, New York,Wiley-Interscience);

[0108] conductivity (see, Anacker (1970) supra; Attwood, D. and R.Natarajan (1979), supra; Moroi, Y., Y. Murata, et al. (1992), supra;Streng, W. H., D. H. -S. Yu, et al. (1996), International Journal ofPharmaceutics 135: 43-52; Iwunze, M. O., M. Lambert, et al. (1997),Monatshefte fur Chemie 128: 582-592; Wiedmer, S. K., M. L. Riekkola, etal. (1997), Analytical Chemistry 69(8): 1577-1584; and Rosen (1978)supra);

[0109] calorimetry (see, Paula, S., W. Sus, et al. (1995), Journal ofPhysical Chemistry 99(30): 11742-11751; Streng, Yu et al. (1996) supra;and Cooper, A., M. A. Nutley, et al. (1998), Analytical Chemistry70(23): 5024-5028);

[0110] ultracentrifugation (see, Anacker (1970) supra; and and Rosen(1978) supra);

[0111] diffusion measurments (see, Anacker (1970) supra; and Jonsson,B., B. Lindman, et al. (1998), SURFACTANTS AND POLYMERS IN AQUEOUSSOLUTION, Chichester, John Wiley Et Sons);

[0112] ultrasonic spectroscopy, densitometry, elastic and quasielasticlight scattering, and small angle neutron scattering, and Brilloum lightscattering (see, D'Arrigo, G., F. Mallamace, et al. (1991), PhysicalReview A 44(4): 2578-2587; and Rosen (1978) supra);

[0113] viscosity measurements (see, Anacker (1970) supra);

[0114] refractive index (see, Anacker (1970) supra; Moroi, Y. et.al.(1990), supra; Moroi, Y., Y. Murata, et al. (1992), supra; and Rosen(1978) supra);

[0115] x-ray diffraction (see, Anacker (1970) supra);

[0116] dye solubilization (see, Anacker (1970) supra; and Rosen (1978)supra);

[0117] fluorescent spectroscopy (see, Iwunze (1997) supra; Rosen (1978)supra; and Jonsson (1998) supra);

[0118] capillary electrophoresis (see, Wiedmer (1997) supra);

[0119] potentiometry, spectrophotometry, and kinetic methods (see,Kopecky, F., Pharmazie 51(3):135-144 (1996);

[0120] osmometry (see, Streng, W. H. et al. (1996) supra; and Rosen(1978) supra); and

[0121] microscopy, optical and non-optical techniques (see, Binks, B.P., Ed. (1999), Modern Characterization Methods of Surfactants Systems,SURFACTANTS SCIENCE SERIES, New York, Marcel Dekker). The disclosures ofthese references as they pertain to methods of measuring aggregation,are hereby incorporated by reference.

[0122] The present invention contemplates, but is not limited by thesespecific examples of techniques for measuring aggregation, and isdefined solely by the claims with other suitable methods of measuringaggregation being contemplated by the present invention.

[0123] The foregoing methods for determining a concentration ofexcipient for combining with the histamine releaser to prepare apharmaceutical formulation are equally applicable to the determinationof a concentration of physiologically acceptable excipient which issufficient to suppress pharmaceutically-induced histamine release froman animal being treated with a histamine releaser. Thus, the presentinvention provides a method for determining a concentration of aphysiologically acceptable excipient that is sufficient to suppresspharmaceutically-induced histamine release in an animal being treatedwith a histamine releaser. The method comprises: a) measuringaggregation of the histamine releaser in a reference solution consistingessentially of the histamine releaser in a concentration at or above thecritical micelle concentration in an aqueous solution; b) measuringaggregation of the histamine releaser in a comparative solutionconsisting essentially of the histamine releaser and a pre-selectedconcentration of the physiologically acceptable excipient in the aqueoussolution, wherein the concentration of the histamine releaser in thecomparative solution is substantially the same as the concentration ofthe histamine releaser in the reference solution; c) optionallyrepeating step b) one or more times with a comparative solution having adifferent pre-selected concentration of the physiologically acceptableexcipient; d) identifying a concentration of physiologically acceptableexcipient that is sufficient to reduce aggregation of the histaminereleaser in the comparative solution by at least about 25 percentcompared to aggregation of the histamine releaser in the referencesolution. The identified concentration of step d) is the concentrationof the physiologically acceptable excipient that is sufficient tosuppress pharmaceutically-induced histamine release in an animal beingtreated with a histamine releaser. The suppression ofpharmaceutically-induced histamine release is discussed further below.The foregoing surface tension and NMR techniques for carrying out themethod of determining a concentration of excipient for combining withthe histamine releaser to prepare a pharmaceutical formulation methodare equally applicable to this method.

[0124] 2.) In vitro Methods

[0125] According to another method of the present invention, theconcentration of excipient is determined by the method comprising thesteps of: a) measuring histamine release from a histamine-containingbiological sample in a reference mixture consisting essentially of: i)the histamine-containing biological sample in a medium and ii) anaqueous solution of the histamine releaser at a concentration sufficientto cause histamine release from the histamine-containing biologicalsample; b) measuring histamine release from the histamine-containingbiological sample in a comparative mixture consisting essentially of: i)the histamine-containing biological sample in medium and ii) an aqueoussolution of the histamine releaser and a pre-selected concentration ofthe physiologically acceptable excipient, wherein the histamine releaserin the comparative mixture is present in a concentration which issubstantially the same as the concentration of histamine releaser in thereference mixture of step a); c) optionally repeating step b) one ormore times with a comparative mixture having a different pre-selectedconcentration of the physiologically acceptable excipient; and d)identifying a concentration of the physiologically acceptable excipientsufficient to reduce histamine release from the histamine-containingbiological sample in the comparative mixture by at least about 10percent compared to histamine release from the histamine-containingbiological sample in the reference mixture. The identified concentrationof step d) is the concentration of physiologically acceptable excipientfor combining with the histamine releaser to prepare the pharmaceuticalformulation.

[0126] The term “histamine containing biological sample” as used hereinincludes cell lines, tissues and biological fluids which include cellsthat contain histamine. Examples of histamine containing biologicalsamples include but are not limited to basophils, mast cells, smoothmuscle cells, cardiac myocytes; reperfused lung, skin, blood vessel,heart, brain or gut tissue; and blood and lymph, which biologicalsamples may be from mammals, including humans, rats, dogs, cats,primates (e.g., monkeys), and the like. Preferably, thehistamine-containing biological sample is selected from the groupconsisting of: human, rat, dog or primate blood; human, rat, dog orprimate basophil cells; and human, rat, dog or primate mast cells. Morepreferably, the histamine-containing biological sample is selected fromthe group consisting of human blood, rat blood, dog blood, rat basophilcells, human mast cells and primate mast cells. The histamine-containingbiological sample is hereinafter sometimes referred to as “biologicalsample” for the sake of brevity.

[0127] The biological sample is provided in medium. When the biologicalsample is cells in culture the medium is the appropriate culture medium.When the biological sample is an isolated tissue or organ in organ bath,the medium is the artificial physiological fluid. When the biologicalsample is blood the medium is plasma. Thus, a suitable medium willdepend upon the particular biological sample employed and will bereadily determined by those skilled in the art based upon conventionalknowledge.

[0128] The order of steps a), b) and c) above is not critical. Thesesteps may be carried out in any desired order, as will be appreciated bythose skilled in the art. It is not necessary that histamine releasefrom the biological sample in the reference mixture be measured beforehistamine release from the biological sample in the comparative solutionis measured, and the present invention contemplates methods comprisingthese steps in any suitable order.

[0129] Step c) of optionally, repeating step b) one or more times meansthat step b) may be repeated with multiple comparative mixtures, but isnot required. One skilled in the art is capable of determining whetherit is desirable in each particular case to carry out step c) (i.e.,repeat step b)) or not. In general, the preferred methods of theinvention include repeating step b) at least once, and more preferablymore than once.

[0130] The reference mixture and comparative mixture(s) can be preparedusing conventional techniques. Generally, the reference mixture andcomparative mixture(s) are prepared by combining or contacting thebiological sample in medium with an aqueous solution. In the case of thereference mixture the aqueous solution contains the histamine releaserat a concentration sufficient to cause measurable histamine release fromthe biological sample when that concentration of histamine releaser iscontacted to or combined with the biological sample in medium. In thecase of the comparative mixture(s), the aqueous solution contains thehistamine releaser and a pre-selected concentration of the excipientwherein the concentration of the histamine releaser in the aqueoussolution, when combined with the biological sample in medium, issufficient to produce a comparative mixture containing substantially thesame concentration of the histamine releaser as in the referencemixture. “Substantially the same” concentration means that thedifference in the concentration of the histamine releaser between thereference mixture and the comparative mixture is not sufficient toproduce a measurable effect on histamine release from the biologicalsample. More preferably, the concentration of the histamine releaser inthe comparative mixture(s) will be considered to be substantially thesame as the concentration in the reference mixture when theconcentration of the comparative mixture(s) is the concentration of thereference mixture plus or minus about 5%. For the most informativecomparison of the histamine release in the reference mixture versushistamine release in the comparative mixture, each of the referencemixture and the comparative mixture(s) will contain an equivalentconcentration of histamine releaser.

[0131] A concentration of the histamine releaser which is sufficient tocause histamine release from the biological sample can be determined bycontacting one or more pre-selected concentrations of histamine releaserto the biological sample in medium and detecting the presence or absenceof histamine release or measuring the amount of histamine released fromthe biological sample. Methods for detecting the presence or absence ofhistamine release and methods for measuring the amount of histaminereleased from the biological sample are discussed further below.

[0132] The aqueous solutions for use in the preparation of the referencemixture and the comparative mixture(s) can be prepared using the samegeneral techniques as described above with reference to methods ofpreparing reference solutions and comparative solutions for analysis ofaggregation. For example, the aqueous solution for use in the referencemixture can be prepared by solubilizing or suspending the histaminereleaser, at a concentration sufficient to cause histamine release fromthe biological sample, in the aqueous solution.

[0133] One or more aqueous solutions for preparation of one or morecomparative mixtures, each including a different pre-selectedconcentration of excipient, can be prepared according to severaldifferent methods. According to one embodiment, aqueous solution(s) forthe comparative mixture(s) are prepared by titrating pre-selected, orotherwise known, concentrations of the excipient into an aqueoussolution containing the appropriate concentration of the histaminereleaser. The excipient may be titrated into the solution containing thehistamine releaser by titrating either up or down, according totechniques known to those skilled in the art.

[0134] According to another embodiment, aqueous solution(s) for thecomparative mixture(s) are prepared by solubilizing a pre-selectedconcentration of excipient in the aqueous solution to create a stocksolution having the highest concentration of excipient, diluting aportion of the stock solution by a suitable dilution factor (e.g.,serially diluting the stock solution) one or more times to preparemultiple aqueous solutions of excipient each containing a pre-selectedconcentration of excipient, and then solubilizing the appropriate amountof histamine releaser in each prepared aqueous solution of excipient sothat when the aqueous solutions are combined with the biological samplein medium to prepare the comparative mixture(s), each comparativemixture will have a histamine releaser concentration substantially thesame as the concentration of histamine releaser in the referencemixture.

[0135] Comparative mixtures containing differing concentrations of acombination of two or more excipients may be utilized in the methods ofthe present invention just as comparative mixtures containing differingconcentrations of only one excipient. It is well within the purview ofone skilled in the art to prepare multiple comparative mixtures, eachcontaining a different pre-selected concentration of each of two or moredifferent excipients. For example, this can be accomplished by preparingmultiple different comparative mixtures each having a differentconcentration of only one of the two or more excipients while theconcentration of the other excipient(s) remain(s) constant.Alternatively, multiple different comparative mixtures can be prepared,each having a different concentration of more than one, or even all ofthe excipients. When the excipient employed in the methods andformulations of the present invention is a combination of two or moreexcipients, all that is required is that the concentration of each ofthe two or more excipients in each comparative mixture should be known.

[0136] Methods of detecting histamine release from the biological sampleand methods for carrying out steps a) and b) of measuring histaminerelease from the biological sample in the reference mixture and thecomparative mixture(s), can be carried out using conventionaltechniques. Examples of conventional techniques for detecting andmeasuring histamine release from a biological sample include thosedescribed in INFLAMMATION: BASIC PRINCIPLES AND CLINICAL CORRELATES(Eds. J. I. Gallin, I. M. Goldstein, and R. Snyderman, Chap. 11,Measurement of Histamine, p. 202, Raven Press, New York, 1992, thesubject matter of which is already incorporated herein by reference inits entirety. Specifically, in vitro and in vivo enzyme-linkedimmunosorbent assay (ELISA) methods and radioimmunoassay (RIA) methodsand fluorometric assay methods may be employed to detect and measurehistamine release from the biological sample. Using ELISA, RIA orfluorometric assay results, the percent inhibition of histamine releasefrom the biological sample can be determined for each of the comparativemixtures relative to histamine release from the biological sample in thereference mixture. Although the foregoing discussion specificallymentions ELISA, RIA and fluorometric assay methods for detectinghistamine release, other conventional methods for determining histaminerelease from a biological sample may be employed.

[0137] The step of identifying the concentration(s) of excipientssufficient to reduce the histamine release from the biological sample byat least about 10 percent comprises identifying the pre-selectedconcentration of the excipient in the comparative mixture(s) which causethe histamine release from the biological sample to be at least about 10percent lower than the histamine release from the biological sample inthe reference mixture. The identified concentration of excipient is theconcentration for combining with the histamine releaser to prepare thepharmaceutical formulations of the invention.

[0138] The foregoing methods for determining a concentration ofexcipient for combining with the histamine releaser to prepare apharmaceutical formulation are equally applicable to the determinationof a concentration of physiologically acceptable excipient which issufficient to suppress pharmaceutically-induced histamine release froman animal being treated with a histamine releaser. Thus, the presentinvention provides a method for determining a concentration of aphysiologically acceptable excipient that is sufficient to suppresspharmaceutically-induced histamine release in an animal being treatedwith a histamine releaser which method comprises: a) measuring histaminerelease from a histamine-containing biological sample in a referencemixture consisting essentially of: i) the histamine-containingbiological sample in a medium and ii) an aqueous solution of thehistamine releaser at a concentration sufficient to cause histaminerelease from the histamine-containing biological sample; b) measuringhistamine release from the histamine-containing biological sample in acomparative mixture consisting essentially of: i) thehistamine-containing biological sample in medium and ii) an aqueoussolution of the histamine releaser and a pre-selected concentration ofthe physiologically acceptable excipient, wherein the histamine releaserin the comparative mixture is present in a concentration which issubstantially the same as the concentration of histamine releaser in thereference mixture of step a); c) optionally repeating step b) one ormore times with a comparative mixture having a different pre-selectedconcentration of the physiologically acceptable excipient; and d)identifying a concentration of the physiologically acceptable excipientsufficient to reduce histamine release from the histamine-containingbiological sample in the comparative mixture by at least about 10percent compared to histamine release from the histamine-containingbiological sample in the reference mixture. The identified concentrationof step d) is the concentration of the physiologically acceptableexcipient that is sufficient to suppress pharmaceutically-inducedhistamine release in an animal being treated with a histamine releaser.

[0139] 3.) In vivo Methods

[0140] Although not the most preferred method, the concentration of theexcipient for use in the present invention can be determined by a methodcomprising the steps of: a) intravenously administering a rapid bolus orrapid infusion of a pharmaceutical formulation comprising atherapeutically effective amount of the histamine releaser and apre-selected concentration of the excipient; b) optionally repeatingstep a) one or more times with a pharmaceutical formulation comprising adifferent pre-selected concentration of the excipient; and c)qualitatively or quantitatively measuring suppression of histaminerelease in vivo as compared to a pharmaceutical formulation containingsubstantially no physiologically acceptable excipient, wherein thesuitable concentration of excipient is the concentration sufficient toqualitatively or quantitatively suppress histamine release in vivo.

[0141] The formulation of step a) can be prepared according to any ofthe methods of the present invention. Step c) of qualitatively orquantitatively measuring suppression of histamine release can be carriedout using the methods described below.

[0142] 4.) Concentrations of Excipients

[0143] The preferred concentrations of excipient(s) varies dependingupon factors such as the category of or the particular excipientemployed and whether the excipient is a combination of two or moreexcipients.

[0144] In general, when the excipient is a divalent inorganic salt, theconcentration of the excipient will be at least about 15 mM, preferablyfrom about 15 mM to about 200 mM, more preferably from about 25 mM toabout 75 mM, regardless of whether the excipient is employed singly orin combination with one or more additional excipients. In the embodimentof the invention wherein the excipient is a single divalent inorganicsalt, preferred concentrations of the excipient will be from about 25 mMto about 75 mM and more preferably about 50 mM. In the embodimentwherein the excipient is calcium chloride, preferred concentrations ofexcipient will be from about 15 mM to about 200 mM, more preferably fromabout 25 mM to about 75 mM, and most preferably about 50 mM.

[0145] When the divalent inorganic salt is employed in combination withone or more additional excipients, the concentration of the divalentinorganic salt may be lower than about 15 mM, particularly as low asabout 10 mM, and is preferably from about 10 mM to about 75 mM and morepreferably from about 25 mM to about 50 mM.

[0146] In general, when the excipient is an organic carboxylic acid, theconcentration of the excipient will be at least about 15 mM, preferablyfrom about 15 mM to about 300 mM, more preferably from about 25 mM toabout 75 mM, regardless of whether the excipient is employed singly orin combination with one or more additional excipients. In the embodimentof the invention wherein the excipient is a single organic carboxylicacid, preferred concentrations of the excipient will be from about 25 mMto about 75 mM and more preferably about 50 mM. In the embodimentwherein the excipient is citric acid, preferred concentrations ofexcipient will be from about 15 mM to about 200 mM, more preferably fromabout 25 mM to about 75 mM, and most preferably about 50 mM.

[0147] When the organic carboxylic acid is employed in combination withone or more additional excipients, the concentration of the organiccarboxylic acid may be lower than about 15 mM, particularly as low asabout 10 mM, and is preferably from about 10 mM to about 100 mM, morepreferably from about 15 mM to about 75 mM and most preferably fromabout 25 mM to about 50 mM.

[0148] In the embodiment wherein the excipient is phosphoric acid, theconcentration of the excipient will typically be from about 6 mM toabout 100 mM, regardless of whether the excipient is employed singly orin combination with one or more additional excipients.

[0149] In general, when the excipient is an amino acid, theconcentration of the amino acid will be at least about 5 mg/mL,preferably from about 10 mg/mL to about 100 mg/mL, more preferably fromabout 10 mg/mL to about 50 mg/mL, and most preferably from about 10mg/mL to about 30 mg/mL, regardless of whether the excipient is employedsingly or in combination with one or more additional excipients. In theembodiment of the invention wherein the excipient is a single aminoacid, preferred concentrations of the excipient will be from about 10mg/mL to about 50 mg/mL, and more preferably between about 10 mg/mL andabout 30 mg/mL, and most preferably about 12.5 mg/mL In particular whenthe excipient is glycine or lysine, preferred concentrations of theexcipient will be from about 10 mg/mL to about 100 mg/mL, morepreferably from about 10 mg/mL to about 30 mg/mL, and most preferablyabout 12.5 mg/mL.

[0150] When the amino acid is employed in combination with one or moreadditional excipients, the concentration of the amino acid may be lowerthan about 5 mg/mL, particularly as low as about 2 mg/mL, and ispreferably from about 5 mg/mL to about 50 mg/mL, more preferably fromabout 10 mg/mL to about 30 mg/mL and most preferably about 12.5 mg/mL

[0151] The concentration of the amino acids is provided in mg/mL unitsfor convenience, however, one skilled in the art can readily calculatethe corresponding concentrations of amino acid in mM units by using themolecular weight of the particular amino acid employed. For example, themolecule weight of glycine is 75, and according a 12.5 mg/mLconcentration of glycine corresponds to 166.5 mM.

[0152] In the embodiment wherein the excipient is a chelating agent, theconcentration of the chelating agent will be at least about 0.02%,preferably from about 0.02% to about 1%, more preferably from about 0.1%to about 0.5%, regardless of whether the excipient is employed singly orin combination with one or more additional excipients. In the embodimentof the invention wherein the excipient is a single chelating agent,preferred concentrations of the excipient will be from about 0.02% toabout 1%, more preferably from about 0.1% to about 0.5%, and mostpreferably about 0.1%. In particular, preferred concentrations of EDTAwill be between about 0.02% and about 1%, more preferably from about0.1% to about 0.5%, and most preferably about 0.1%. Percentages arebased upon weight unless otherwise indicated.

[0153] When employed in combination with one or more additionalexcipients, the concentration of the chelating agents are preferablyfrom about 0.05% to about 1% and more preferably about 0.1%.

[0154] The concentration of the chelating agents is provided in % unitsfor convenience, however, one skilled in the art can readily calculatethe corresponding concentrations of chelating agents in mM units byusing the molecular weight of the particular chelating agent employed.For example, the molecule weight of EDTA (free acid) is 292.2, andaccordingly a 0.1% concentration of EDTA corresponds to 2.69 mM.

[0155] In the embodiment wherein the excipient is albumin, theconcentration of the excipient will typically be from about 1 mg/mL,preferably from about 1 mg/mL to about 25 mg/mL, more preferably fromabout 5 mg/mL to about 15 mg/mL, and most preferably about 10 mg/mL,regardless of whether the excipient is employed singly or in combinationwith one or more additional excipients.

[0156] To further illustrate specific concentrations of the excipient(s)according to the present invention, the following embodiments includingtwo or more excipients are provided. In one embodiment, the excipient isa combination of citric acid in a concentration of not less than about15 mM, preferably from about 15 mM to about 100 mM and more preferablyabout 50 mM, and EDTA in a concentration of from about 0.02% to about1%, preferably from about 0.1% to about 0.5% and more preferably about0.1%. In one preferred embodiment, the excipient is a combination ofcitric acid in a concentration of not less than about 15 mM, preferablyfrom about 15 mM to about 100 mM and more preferably about 50 mM, andcalcium chloride in a concentration of not less than about 15 mM,preferably from about 25 mM to about 75 mM and more preferably about 50mM. In one embodiment, the excipient is a combination of citric acid ina concentration of not less than about 15 mM, preferably from about 15mM to about 100 mM and more preferably about 50 mM, and glycine in aconcentration of from about 10 mg/mL to about 100 mg/mL, preferably fromabout 10 mg/mL to about 30 mg/mL and more preferably about 12.5 mg/mL.In one embodiment the excipient is a combination of citric acid in aconcentration of not less than about 15 mM, preferably from about 15 mMto about 100 mM and more preferably about 50 mM, glycine in aconcentration of from about 10 mg/mL to about 100 mg/mL, preferably fromabout 10 mg/mL to about 30 mg/mL and more preferably about 12.5 mg/mL,and EDTA in a concentration of from about 0.02% to about 1%, preferablyfrom about 0.1% to about 0.5% and more preferably about 0.1%. Otherspecific examples of combinations of excipients will be readilydeterminable by those skilled in the art based upon the foregoingdescription for determining appropriate concentrations and thesespecific examples.

[0157] D. Combining the Histamine Releaser and the Excipient

[0158] The step of combining a therapeutically effective amount of thehistamine releaser with the concentration of excipient may be carriedout by any suitable means known to those skilled in the art. Forexample, the histamine releaser and excipient may be admixed in solidphase. Alternatively, either the histamine releaser or the excipient maybe solubilized or suspended in a suitable physiologically acceptablediluent, and the other component may be added thereto and solubilized orsuspended in the diluent. In one preferred embodiment, the excipient issolubilized in a physiologically acceptable diluent and the histaminereleaser is added to the solution containing the excipient solubilizedtherein. If desired for optimization of the formulation (as describedbelow), the pH of the solution containing the one component in diluent,i.e., either the excipient or the histamine releaser, may be adjustedprior to the addition of the other component. In one preferredembodiment, the histamine releaser and excipient are combined bysolubilizing the excipient in a physiologically acceptable diluent,adjusting pH and then adding the histamine releaser to the diluent andsolubilizing therein.

[0159] E. Methods for Optimizing the Formulation with pH

[0160] Aggregation of the histamine releaser and histamine release invivo may be affected in some circumstances by pH. The pH of theformulation containing the histamine releaser can also play a role inthe charge, ionic state or solvation capabilities of the histaminereleaser In some cases, pH can play a beneficial role in the physicaland/or chemical stabilization of certain histamine releasers insolution. For example, certain non-steroidal neuromuscular blockers,such as Compound 1, are known to be more chemically stable at acidic pH,preferably a pH of between about 2 and about 5. For these reasons, itmay be desirable or advantageous in some cases to evaluate comparativesolutions and mixtures of differing pH, for the purpose of optimizingthe pharmaceutical formulations according to the present invention, andfor maximizing the suppression of pharmaceutically-induced histaminerelease.

[0161] Thus, according to one embodiment of the present invention thepharmaceutical formulation is optimized for pH. The method foroptimizing the pharmaceutical formulation for pH comprises the steps of:a) measuring aggregation of the histamine releaser in a referencesolution consisting essentially of the histamine releaser in aconcentration at or above the critical micelle concentration in anaqueous solution; b) measuring aggregation of the histamine releaser ina comparative solution consisting essentially of the histamine releaserand a pre-selected or pre-determined concentration of thephysiologically acceptable excipient in the aqueous solution, whereinthe concentration of the histamine releaser in the comparative solutionis substantially the same as the concentration of the histamine releaserin the reference solution, and wherein the comparative solution has apre-selected pH; c) optionally repeating step b) one or more times witha comparative solution having substantially the same or a differentpre-selected concentration of physiologically acceptable excipient and adifferent pre-selected pH, and d) identifying the pH of the comparativesolution which provides the optimum reduction in aggregation of thehistamine releaser in the comparative solution. The identified pH ofstep d) is the pH for preparing an optimized pharmaceutical formulationaccording to the present invention.

[0162] According to another embodiment the pharmaceutical formulationprepared according to the methods of the present invention is optimizedfor pH using the method comprising the steps of: a) measuring histaminerelease from a histamine-containing biological sample in a referencemixture consisting essentially of: i) the histamine-containingbiological sample in a medium and ii) an aqueous solution of thehistamine releaser at a concentration sufficient to cause histaminerelease from the histamine-containing biological sample; b) measuringhistamine release from the histamine-containing biological sample in acomparative mixture consisting essentially of: i) thehistamine-containing biological sample in medium and ii) an aqueoussolution of the histamine releaser and a pre-selected concentration ofthe physiologically acceptable excipient, wherein the histamine releaserin the comparative mixture is present in a concentration which issubstantially the same as the concentration of histamine releaser in thereference mixture of step a) and wherein the comparative mixture has apre-selected pH; c) optionally repeating step b) one or more times witha comparative mixture having substantially the same or a differentpre-selected concentration of physiologically acceptable excipient and adifferent pre-selected pH, and d) identifying the pH of the comparativemixture which provides the optimum reduction of histamine release fromthe histamine-containing biological sample in the comparative mixture.The identified pH of step d) is the pH for preparing an optimizedpharmaceutical formulation according to the present invention.

[0163] Based upon the preceding examples, one skilled in the art canadapt the teachings above to optimize the pharmaceutical formulations ofthe present invention regardless of the method which is employed fordetermining the concentration of excipient. The present inventionexpressly contemplates using such methods to optimize the pH of thepharmaceutical formulations of the present invention.

[0164] The optimum pharmaceutical formulation though, will also takeinto account the effect of the pH of the formulation on the stability ofthe particular histamine releaser in the formulation. As noted abovesome histamine releasers are more physically and/or chemically stable atan acidic pH. Other histamine releasers may be more physically and/orchemically stable at a basic pH.

[0165] As will be apparent to those skilled in the art, thepharmaceutical formulations prepared according to the methods of thepresent invention may be adjusted for pH by titrating formulations ofhistamine releaser and physiologically acceptable excipient(s) with anagent suitable for adjusting pH. Agents suitable for adjusting pH willbe apparent to those skilled in the art and can include for exampleacids, bases, pH buffers and salts.

[0166] Typically, the pH of the optimized pharmaceutical formulations ofthe present invention will be adjusted as necessary to obtain a pH ofthe pharmaceutical formulation which is between about 2 and about 10. Inone preferred embodiment the pH of the optimized pharmaceuticalformulation will be between about 2 and about 8. In one preferredembodiment, the pH of the optimized pharmaceutical formulation will bebetween about 2 and about 5, more preferably between about 2 and about4. In the embodiment of the present invention wherein the histaminereleaser is Compound 1, the optimized pharmaceutical formulation willtypically have a pH of from about 2 to about 5, preferably from about 2to about 4, and more preferably about 3.

[0167] IV. Formulations

[0168] The pharmaceutical formulations prepared according to any of theforegoing methods of the present invention may include only thetherapeutically effective amount of the histamine releaser and theconcentration of the physiologically acceptable excipient. However, inpreferred embodiments, the pharmaceutical formulations of the inventionalso include a physiologically acceptable diluent or vehicle. Thediluent facilitates the delivery of the histamine releaser and theexcipient to the animal being treated therewith. The selection of asuitable diluent will depend upon the type of pharmaceutical formulation(e.g., solution, dispersion, emulsion, etc.), and is readily determinedby those skilled in the art of pharmaceutical sciences.

[0169] The pharmaceutical formulations of the invention are in a formsuitable for parenteral administration, and preferably in a formsuitable for intravenous administration. Formulations suitable forintravenous administration include aqueous sterile injection solutions,aqueous and non-aqueous sterile suspensions, and sterile emulsions.

[0170] Aqueous sterile injection solutions typically comprise thehistamine releaser and excipient in a physiologically acceptable diluentsuch as water for injection, sodium chloride for injection, or dextrosefor injection. The sterile injection solutions may also contain otherphysiologically acceptable additives such as other acids (e.g.,hydrochloric acid) and bases (e.g., sodium hydroxide) for pH adjustment,pH buffers, and co-solvents. Examples of suitable co-solvents which canbe employed in the formulations of the present invention include but arenot limited to ethanol, propylene glycol, benzyl alcohol andcombinations thereof. Other suitable additives will be apparent to thoseskilled in the art. Sterile injection solutions may be prepared usingconventional techniques of pharmaceutical sciences.

[0171] Aqueous and non-aqueous sterile suspensions can include, inaddition to the histamine releaser, excipient, diluent, and additivespreviously mentioned, suspending agents and thickening agents, andliposomes or other microparticulate systems. Non-aqueous sterilesuspensions, may employ water, parabens, glycerol, soybean oil,safflower oil, and the like, and also combinations thereof as thediluent. Suspensions may be prepared using techniques known the in artof pharmaceutical sciences.

[0172] Sterile emulsions can include oil-in-water emulsions andwater-in-oil emulsions. Emulsions may include glycerol, soybean oil,safflower oil, and the like and combinations thereof as the oil phase.Sterile emulsions may be prepared using techniques known the in art ofpharmaceutical sciences. Emulsions may include the other additivespreviously mentioned as well.

[0173] The formulations may also be presented as lyophilized solids forreconstitution. Such lyophilized formulations are typicallyreconstituted with water for injection, sodium chloride for injection ordextrose solution. Lyophilized formulations may include conventionallyophilization bulking agents such as β-cyclodextrin and lactose. Suchformulations are typically presented in unit dosage forms such as vialsor disposable injection devices. They may also be presented inmulti-dose forms such as a bottle from which the appropriate dose may bewithdrawn. All such formulations should be sterile.

[0174] The appropriate dosage of histamine releaser for inclusion in theformulations of the invention will depend upon the particular histaminereleaser and the desired therapeutic effect. Advantageously,conventional dosages of these agents can be employed in the methods andformulations of the present invention.

[0175] Preferred formulations of the present invention include aneuromuscular blocker, most preferably Compound 1, as the histaminereleaser and one or more excipients, in a form suitable for intravenousadministration. Preferred excipients for use in the formulations of thepresent invention may be selected from the group consisting of divalentinorganic salts, organic carboxylic acids, phosphoric acid, amino acids,chelating agents, albumins and combinations thereof. More preferably,the formulation also includes a diluent selected from the groupconsisting of water for injection, sodium chloride for injection anddextrose solutions. The concentration of neuromuscular blocker istypically from about 1 mM to about 55 mM. The appropriate concentrationof excipient is determined according to the preceding methods.

[0176] In one preferred embodiment, the formulation comprises atherapeutically effective amount of(Z)-2-chloro-1-{3-{(1R,2S)-6,7-dimethoxy-2-methyl-1-[(3,4,5-trimethoxyphenyl)methyl]-1,2,3,4-tetrahydro-2-isoquinolinio}propyl}-4-{3-[(1S,2R)-6,7-dimethoxy-2-methyl-1-(3,4,5-trimethoxyphenyl)-1,2,3,4-tetrahydro-2-isoquinolinio]propyl}-2-butenedioatedichloride or a pharmaceutically acceptable salt thereof, together withcitric acid in a concentration of from about 25 to about 75 mM, mostpreferably about 50 mM.

[0177] In one embodiment, the formulation comprises a therapeuticallyeffective amount of(Z)-2-chloro-1-{3-{(1R,2S)-6,7-dimethoxy-2-methyl-1-[(3,4,5-trimethoxyphenyl)methyl]-1,2,3,4-tetrahydro-2-isoquinolinio}propyl}-4-{3-[(1S,2R)-6,7-dimethoxy-2-methyl-1-(3,4,5-trimethoxyphenyl)-1,2,3,4-tetrahydro-2-isoquinolinio]propyl}-2-butenedioatedichloride or a pharmaceutically acceptable salt thereof, together withEDTA in a concentration of from about 0.1% to about 0.5%, mostpreferably about 0.1%.

[0178] In one embodiment, the formulation comprises a therapeuticallyeffective amount of(Z)-2-chloro-1-{3-{(1R,2S)-6,7-dimethoxy-2-methyl-1-[(3,4,5-trimethoxyphenyl)methyl]-1,2,3,4-tetrahydro-2-isoquinolinio}propyl}-4-{3-[(1S,2R)-6,7-dimethoxy-2-methyl-1-(3,4,5-trimethoxyphenyl)-1,2,3,4-tetrahydro-2-isoquinolinio]propyl}-2-butenedioatedichloride or a pharmaceutically acceptable salt thereof, together witha combination of citric acid in a concentration of from about 25 toabout 75 mM, most preferably about 50 mM, and EDTA in a concentration offrom about 0.1% to about 0.5%, most preferably about 0.1%.

[0179] In one embodiment, the formulation comprises a therapeuticallyeffective amount of(Z)-2-chloro-1-{3-{(1R,2S)-6,7-dimethoxy-2-methyl-1-[(3,4,5-trimethoxyphenyl)methyl]-1,2,3,4-tetrahydro-2-isoquinolinio}propyl}-4-{3-[(1S,2R)-6,7-dimethoxy-2-methyl-1-(3,4,5-trimethoxyphenyl)-1,2,3,4-tetrahydro-2-isoquinolinio]propyl}-2-butenedioatedichloride or a pharmaceutically acceptable salt thereof, together withcalcium chloride in a concentration of from about 25 to about 75 mM,most preferably about 50 mM.

[0180] In one embodiment, the formulation comprises a therapeuticallyeffective amount of(Z)-2-chloro-1-{3-{(1R,2S)-6,7-dimethoxy-2-methyl-1-[(3,4,5-trimethoxyphenyl)methyl]-1,2,3,4-tetrahydro-2-isoquinolinio}propyl}-4-{3-[(1S,2R)-6,7-dimethoxy-2-methyl-1-(3,4,5-trimethoxyphenyl)-1,2,3,4-tetrahydro-2-isoquinolinio]propyl}-2-butenedioatedichloride or a pharmaceutically acceptable salt thereof, together witha combination of citric acid in a concentration from about 25 to about75 mM, most preferably about 50 mM, and calcium chloride in aconcentration from about 25 to about 75 mM, most preferably about 50 mM.

[0181] In one embodiment, the formulation comprises a therapeuticallyeffective amount of(Z)-2-chloro-1-{3-{(1R,2S)-6,7-dimethoxy-2-methyl-1-[(3,4,5-trimethoxyphenyl)methyl]-1,2,3,4-tetrahydro-2-isoquinolinio}propyl}-4-{3-[(1S,2R)-6,7-dimethoxy-2-methyl-1-(3,4,5-trimethoxyphenyl)-1,2,3,4-tetrahydro-2-isoquinolinio]propyl}-2-butenedioatedichloride or a pharmaceutically acceptable salt thereof, together withglycine in a concentration of from about 10 mg/mL to about 30 mg/mL,preferably about 12.5 mg/mL.

[0182] In one embodiment, the formulation comprises a therapeuticallyeffective amount of(Z)-2-chloro-1-{3-{(1R,2S)-6,7-dimethoxy-2-methyl-1-[(3,4,5-trimethoxyphenyl)methyl]-1,2,3,4-tetrahydro-2-isoquinolinio}propyl}-4-{3-[(1S,2R)-6,7-dimethoxy-2-methyl-1-(3,4,5-trimethoxyphenyl)-1,2,3,4-tetrahydro-2-isoquinolinio]propyl}-2-butenedioatedichloride or a pharmaceutically acceptable salt thereof, together witha combination of glycine in a concentration of from about 10 mg/mL toabout 30 mg/mL, preferably about 12.5 mg/mL, and EDTA in a concentrationof from about 0.1% to about 0.5%, most preferably about 0.1%.

[0183] In one embodiment, the formulation comprises a therapeuticallyeffective amount of(z)-2-chloro-1-{3-{(1R,2S)-6,7-dimethoxy-2-methyl-1-[(3,4,5-trimethoxyphenyl)methyl]-1,2,3,4-tetrahydro-2-isoquinolinio}propyl}-4-{3-[(1S,2R)-6,7-dimethoxy-2-methyl-1-(3,4,5-trimethoxyphenyl)-1,2,3,4-tetrahydro-2-isoquinolinio]propyl}-2-butenedioatedichloride or a pharmaceutically acceptable salt thereof, together witha combination of glycine in a concentration of from about 10 mg/mL toabout 30 mg/mL, preferably about 12.5 mg/mL, and citric acid in aconcentration of from about 25 to about 75 mM, most preferably about 50mM.

[0184] In one embodiment, the formulation comprises a therapeuticallyeffective amount of(Z)-2-chloro-1-{3-{(1R,2S)-6,7-dimethoxy-2-methyl-1-[(3,4,5-trimethoxyphenyl)methyl]-1,2,3,4-tetrahydro-2-isoquinolinio}propyl}-4-{3-[(1S,2R)-6,7-dimethoxy-2-methyl-1-(3,4,5-trimethoxyphenyl)-1,2,3,4-tetrahydro-2-isoquinolinio]propyl}-2-butenedioatedichloride or a pharmaceutically acceptable salt thereof, together witha combination of glycine in a concentration of from about 10 mg/mL toabout 30 mg/ml, preferably about 12.5 mg/mL, citric acid in aconcentration of from about 25 to about 75 mM, most preferably about 50mM, and EDTA in a concentration of from about 0.1% to about 0.5%, mostpreferably about 0.1%. Particularly preferred of the foregoing specificexamples of pharmaceutical formulations are formulations having a pHbetween about 2 and about 5, more preferably between about 2 and about 4and most preferably about 3. In the formulations noted above, pHadjustment to the ranges noted may or may not be necessary dependingupon the particular excipients and concentrations employed.

[0185] V. Methods for Suppressing Histamine Release

[0186] The present invention also provides methods for suppressingpharmaceutically-induced histamine release in an animal being treatedwith a histamine releaser. “Pharmaceutically-induced histamine release”as used herein refers to histamine release in vivo which is induced orat least partially caused by the intravenous rapid bolus or rapidinfusion administration of a histamine releaser.Pharmaceutically-induced histamine release is described in Goodman

Gilman's The PHARMACOLOGICAL BASIS OF THERAPEUTICS, 9th ed. McGraw-Hill,New York, (1996) pp. 581-593.

[0187] As used herein, to “suppress pharmaceutically-induced histaminerelease” means to substantially completely prevent histamine release orreduce the amount or the rate of histamine release fromhistamine-containing cells, tissues or fluids in vitro upon exposure toa histamine releaser; or to substantially completely prevent histaminerelease, or reduce the amount or the rate of histamine release fromhistamine-containing cells, tissues or fluids in vivo uponadministration of a formulation containing a histamine releaser to ananimal. More specifically, “suppression of pharmaceutically-inducedhistamine release” in vivo refers to suppression of histamine release invivo upon intravenous administration of a histamine releaser as a rapidbolus or rapid infusion. Suppression of pharmaceutically-inducedhistamine release can be observed qualitatively and/or quantitatively.

[0188] Pharmaceutically-induced histamine release can be qualitativelyobserved by intravenously administering a rapid bolus or rapid infusionof the histamine releaser to the animal and observing the physicalmanifestations described above, which are associated with elevatedplasma and/or tissue concentrations of histamine. Suppression ofpharmaceutically-induced histamine release, therefore is qualitativelyobserved by intravenously administering to the animal being treated, arapid bolus or rapid infusion of any of the pharmaceutical formulationsof the present invention and observing comparatively less severephysiological manifestations, or even an absence of the physiologicalmanifestations, associated with elevated plasma and/or tissue histaminelevels.

[0189] Suppression of pharmaceutically-induced histamine release can bequantitatively observed by intravenously administering to the animalbeing treated a rapid bolus or rapid infusion of the pharmaceuticalformulation according to the present invention and observing, andmeasuring the plasma and/or tissue histamine levels present in theanimal subsequent to administration. Plasma histamine levels present inthe animal subsequent to administration can be measured by withdrawing ablood sample from the animal after administration of the pharmaceuticalformulation. The blood sample is preferably withdrawn from the region ator near the site of administration of the pharmaceutical formulation.Tissue histamine levels present in the animal subsequent toadministration can be measured by taking a tissue sample from the animalafter administration of the pharmaceutical formulation. The sample ispreferably taken from the region at or near the site of administrationof the pharmaceutical formulation. Histamine levels present in eitherthe plasma or tissue sample can be measured using the techniquesdescribed above for detecting histamine release from a biologicalsample, e.g., ELISA, RIA and fluorometric assays.

[0190] In general, the methods for suppressing pharmaceutically-inducedhistamine release in an animal being treated with a histamine releasercomprise administering to the animal, any pharmaceutical formulationaccording to the present invention. In preferred methods, thepharmaceutical formulation will include an excipient selected from thegroup consisting of divalent inorganic salts, organic carboxylic acids,phosphoric acid, amino acids, chelating agents, albumins andcombinations thereof.

[0191] The step of administering comprises parenterally administeringthe pharmaceutical formulation to the animal, more preferably,intravenously administering the pharmaceutical formulation, and mostpreferably intravenously administering the pharmaceutical formulation asa rapid bolus or rapid infusion. The formulations of the presentinvention advantageously permit intravenous administration as a rapidbolus or rapid infusion, while suppressing the concomitant release ofhistamine that is typically observed with administration of conventionalformulations of histamine releasers by this technique.

[0192] More specifically, one method for suppressingpharmaceutically-induced histamine release in an animal being treatedwith a histamine releaser comprises administering to an animal in needthereof, a pharmaceutical formulation comprising a therapeuticallyeffective amount of the histamine releaser and a concentration ofphysiologically acceptable excipient which concentration, when combinedin an aqueous solution with the histamine releaser at or above thecritical micelle concentration, is sufficient to reduce aggregation ofthe histamine releaser in the aqueous solution by at least about 25percent compared to aggregation of the histamine releaser in an aqueoussolution containing substantially no physiologically acceptableexcipient.

[0193] Another method for suppressing pharmaceutically-induced histaminerelease in an animal being treated with a histamine releaser comprisesadministering to an animal in need thereof, a pharmaceutical formulationcomprising a therapeutically effective amount of the histamine releaserand a concentration of physiologically acceptable excipient whichconcentration, is determined by the method comprising the steps of: a)measuring aggregation of the histamine releaser in a reference solutionconsisting essentially of the histamine releaser in a concentration ator above the critical micelle concentration in an aqueous solution; b)measuring aggregation of the histamine releaser in a comparativesolution consisting essentially of the histamine releaser and apre-selected concentration of the physiologically acceptable excipientin the aqueous solution, wherein the concentration of the histaminereleaser in the comparative solution is substantially the same as theconcentration of the histamine releaser in the reference solution; c)optionally repeating step b) one or more times with a comparativesolution having a different pre-selected concentration of thephysiologically acceptable excipient; d) identifying a concentration ofphysiologically acceptable excipient that is sufficient to reduceaggregation of the histamine releaser in the comparative solution by atleast about 25 percent compared to aggregation of the histamine releaserin the reference solution; wherein the identified concentration of stepd) is the concentration of the physiologically acceptable excipient thatis sufficient to suppress pharmaceutically-induced histamine release inan animal being treated with a histamine releaser and is theconcentration of physiologically acceptable excipient for combining withthe histamine releaser to prepare the pharmaceutical formulation foradministration to the animal being treated therewith.

[0194] Another method for suppressing pharmaceutically-induced histaminerelease in an animal being treated with a histamine releaser comprisesadministering to an animal in need thereof, a pharmaceutical formulationcomprising a therapeutically effective amount of the histamine releaserand a concentration of physiologically acceptable excipient whichconcentration, is determined by the method comprising the steps of: a)measuring histamine release from a histamine-containing biologicalsample in a reference mixture, b) measuring histamine release from thehistamine-containing biological sample in a comparative mixture, c)optionally repeating step b) one or more times with a comparativemixture having a different pre-selected concentration of thephysiologically acceptable excipient; and d) identifying a concentrationof the physiologically acceptable excipient sufficient to reducehistamine release from the histamine-containing biological sample in thecomparative mixture by at least about 10 percent compared to histaminerelease from the histamine-containing biological sample in the referencemixture; wherein the identified concentration of step d) is theconcentration of physiologically acceptable excipient for combining withthe histamine releaser to prepare the pharmaceutical formulation. Thereference mixture consists essentially of: i) the histamine-containingbiological sample in a medium and ii) an aqueous solution of thehistamine releaser at a concentration sufficient to cause histaminerelease from the histamine-containing biological sample. The comparativemixture consists essentially of: i) the histamine-containing biologicalsample in medium and ii) an aqueous solution of the histamine releaserand a pre-selected concentration of the physiologically acceptableexcipient, wherein the histamine releaser in the comparative mixture ispresent in a concentration which is substantially the same as theconcentration of histamine releaser in the reference mixture of step a).

[0195] Histamine release in vivo may be triggered differently among andeven within animal species (e.g., different sites may be involved anddifferent animals may have different sensitivities). Accordingly themethods of treatment may be optimized by one skilled in the art, for aparticular species or individual subject. The methods of treatment areuseful for the treatment of a variety of animals, preferably mammals,including humans, dogs, and primates (e.g., monkeys) and most preferablyhumans.

[0196] The present invention also comprises methods for preventingcardiovascular and respiratory effects mediated bypharmaceutically-induced histamine release in an animal being treatedwith a histamine-releaser. “Cardiovascular and respiratory effectsmediated by pharmaceutically-induced histamine release” as used hereinrefers to cardiovascular and respiratory effects induced by elevatedplasma and/or tissue histamine levels, i.e., plasma or tissue histaminelevels above normal physiological levels. Such histamine-inducedcardiovascular and respiratory effects can include, but are not limitedto flushing, hypotension, tachycardia, bronchoconstriction,anaphylactoid reactions and anaphylactic shock, and combinations of anytwo or more of the foregoing.

[0197] The terms “prevent” or “prevention” as used herein with referenceto a particular condition refers to a decrease in the incidence and/orseverity of the condition, as well as avoidance of the condition.

[0198] The method for preventing cardiovascular effects mediated bypharmaceutically-induced histamine release in an animal being treatedwith a histamine-releaser comprises administering to the animal, anypharmaceutical formulation according to the present invention. Bysuppressing the histamine release typically induced by the intravenousrapid bolus or rapid infusion administration of a histamine releaser,the undesirable cardiovascular effects resulting from elevated plasmaand tissue histamine levels are prevented.

[0199] VI. Kits

[0200] The present invention also includes a kit useful for thepreparation of pharmaceutical formulations of histamine releasersaccording to the present invention. The kit comprises: a) aphysiologically acceptable excipient, and b) instructions for preparingthe pharmaceutical formulation according to the present invention.

[0201] The excipient is preferably selected from the group consisting ofdivalent inorganic salts, organic carboxylic acids, phosphoric acid,amino acids, chelating agents, albumins and combinations thereof. Morepreferably, the physiologically acceptable excipient is selected fromthe group consisting of calcium chloride, sodium sulfate, magnesiumsulfate, tartaric acid, maleic acid, acetic acid, citric acid, succinicacid, glucuronic acid, phosphoric acid, glycine, lysine, arginine, EDTA,bovine serum albumin, human serum albumin and combinations thereof.

[0202] The instructions include instructions for combining the histaminereleaser with the concentration of the excipient in order to prepare anyof the pharmaceutical formulations of the present invention. Morespecifically, the instructions can include instructions for combiningthe histamine releaser with a concentration of the excipient which issufficient, when combined in an aqueous solution with the histaminereleaser at or above critical micelle concentration, to reduceaggregation of the histamine releaser in the aqueous solution by atleast about 25 percent compared to aggregation of the histamine releaserin the aqueous solution containing substantially no physiologicallyacceptable excipient. The instructions can include instructions forcombining the histamine releaser with the excipient in concentrationssufficient to suppress pharmaceutically-induced histamine release fromthe animal being treated with the histamine releaser.

[0203] In a preferred embodiment, the instructions will include theprecise amount of histamine releaser required to achieve the desiredtherapeutic, diagnostic or medicinal effect, which amount will bespecific for the particular histamine releaser employed. Thus, theinstructions may include specific amounts for several different specifichistamine releasers, which specific amount for each specific histaminereleaser is a therapeutically effective amount.

[0204] In another preferred embodiment, the instructions will includethe precise amount of excipient for combining with the histaminereleaser, which amount will be specific for the particular excipient orcombination of excipients for use with the particular histamine releaserwith which the excipient will be combined to prepare the pharmaceuticalformulation. The precise amount of excipient provided, will have beenpre-determined using any one or more of the methods of the presentinvention for determining the concentration of excipient for combiningwith the histamine releaser to prepare the pharmaceutical formulation.

[0205] The kit may also include the histamine releaser. In thisembodiment, the instructions include instructions for preparing apharmaceutical formulation according to the present invention bycombining a therapeutically effective amount of the provided histaminereleaser with a concentration of excipient that is sufficient tosuppress pharmaceutically induced histamine release from the animalbeing treated with the histamine releaser and to whom the preparedformulation will be administered. In one preferred embodiment, the kitincludes the histamine releaser,(Z)-2-chloro-1-{3-{(1R,2S)-6,7-dimethoxy-2-methyl-1-[(3,4,5-trimethoxyphenyl)methyl]-1,2,3,4-tetrahydro-2-isoquinolinio}propyl}-4-{3-[(1S,2R)-6,7-dimethoxy-2-methyl-1-(3,4,5-trimethoxyphenyl)-1,2,3,4-tetrahydro-2-isoquinolinio]propyl}-2-butenedioate dichloride or a pharmaceutically acceptable saltthereof.

[0206] Depending upon the particular histamine releaser and excipientsemployed in the preparation of the pharmaceutical formulation, theinstructions may also include instructions for adjusting pH of thepharmaceutical formulation to from about 2 to about 8, preferably fromabout 2 to about 5 and more preferably to about 3.

[0207] The kit may also include additional components. For example, thekit may also include a physiologically acceptable diluent forsolubilizing and/or reconstituting the histamine releaser and thephysiologically acceptable excipient. In this embodiment, theinstructions include instructions for combining the histamine releaserand the physiologically acceptable excipient in the diluent. As anotherexample, the kit may include one or more containers for combining thehistamine releaser and the excipient.

[0208] The following examples are intended for illustration only and arenot intended to limit the scope of the invention, the invention beingdefined by the claims.

[0209] As used herein: “mM” means millimolar; “nM” means nanomolar;“mg/mL” means milligrams per milliliter; “%” means percent by weight;“NaCl” means sodium chloride; “D2O” means deuterated water; “D2O saline”means deuterated saline solution; “NaOD” means deuterated sodiumhydroxide; “DCl” means deuterated hydrochloric acid; “d₄-citrate” meansdeuterated citric acid; “CaCl₂” means calcium chloride; “pD” means pHuncorrected for the deuterium isotope effect; “μL” means microliters;“μm” means micrometer; “MHz” means megahertz; “ppm” means parts permillion; “° C.” means degrees Centigrade; “mmHg” means millimeters ofmercury; and “Compound 1” means(Z)-2-chloro-1-{3-{(1R,2S)-6,7-dimethoxy-2-methyl-1-[(3,4,5-trimethoxyphenyl)methyl]-1,2,3,4tetrahydro-2-isoquinolinio}propyl}-4-{3-[(1S,2R)-6,7-dimethoxy-2-methyl-1-(3,4,5-trimethoxyphenyl)-1,2,3,4-tetrahydro-2-isoquinolinio]propyl}-2-butenedioatedichloride or a pharmaceutically acceptable salt thereof.

EXAMPLE 1

[0210] Surface Tension Analysis

[0211] The surface tension properties of the histamine liberating agentcan be evaluated using the DuNouy ring method. This and other methodsfor evaluating surface tension properties are described in theliterature. See, PHYSICAL PHARMACY: PHYSICAL CHEMICAL PRINCIPLES IN THEPHARMACEUTICAL SCIENCES, Editors: Alfred Martin, James Swarbrick, ArthurCammarata, Third Edition, Lea Et Febiger, Philadelphia, 1983.

[0212] The DuNuoytensiometer, commercially available from Kruss GMBH,Hamburg Germany, is used for measuring the surface and interfacialtension of liquids. The principle of the instrument depends on the factthat the force necessary to detach a platinum-iridium ring immersed atthe surface is proportional to the surface tension.

[0213] The force required to detach the ring in this manner is providedby a torsion wire and is recorded in dynes on a calibrated dial. Thesurface tension is given by the formula:$\mathrm{\Upsilon} = \frac{{dial}\quad {reading}\quad {in}\quad {dynes}}{2 \times {ring}\quad {circumference} \times {correction}\quad {factor}}$

[0214] where the ring circumference and the correction factor areequipment specific.

[0215] The surface tension of most liquids decreases almost linearlywith an increase in temperature, and thus it is necessary to control thetemperature of the system when carrying out surface tensiondeterminations.

[0216] The tensiometer is calibrated with filtered water and the surfacetension of a 50 mg/mL solution of Compound 1 in water is evaluated atroom temperature. The solution is diluted to obtain solutions having thefollowing concentrations (mg/mL) of Compound 1: 47.5, 45, 42.5, 40,37.5, 35, 32.5, 30, 27.5, 25, 22.5, 20, 17.5, 15, 12.5, 10, 5,3,2, and1.

[0217] The surface tension of each solution is evaluated at roomtemperature and the results are recorded in a curve of surface tension(y-axis) versus concentration of drug α-axis). The results are reportedin FIG. 1. FIG. 1 indicates that Compound 1 is surface active attherapeutically effective doses and reveals concentration dependentsurface tension lowering.

[0218] Referring to FIG. 1, the change in slope of the surface tensiongraph with increasing concentration of Compound 1, which occurs atapproximately 15-17.5 mg/mL indicates the critical micelle concentrationof Compound 1 in water.

EXAMPLE 2

[0219] Proton NMR Analysis

[0220] A. General Procedures

[0221] Compound 1 and excipient (in the various amounts noted below) aredissolved in 200 μL of D₂O with 0.15 M NaCl adjusted to pD 3.0 with NaODand/or DCl for compound stability reasons. Deuterated saline solution isused in the reference solution because critical compound peaks (orsignals) overlap when deuterated water is employed. Samples in atitration series are prepared by serial dilutions. All NMR spectra arerecorded at 30° C. on a Varian Unity Plus 500 MHz spectrometer. Spectraare referenced to D₂O at 4.65 ppm. All spectra are processed usingVarian VNMR® software v6.1a. T₁ and T₂ relaxation rates are measuredusing the standard inversion-recovery and Carr-Purcell-Meiboom-Gill(CPMG) experiments respectively, both of which are provided with theVarian software. CPMG experiments are described in M. Bulsing, et al.,J. Chem. Soc. Chem. Commun. 1201-3 (1981); R. Freeman et al., IN DYNAMIcNMR SPECTROSCOPY, eds. L. Jackman and F. Cotton (1975) pp. 131-162,Academic Press, London and New York; and D. Rabenstein, et al., J. Magn.Reson. 64:541-546 (1985).

[0222] B. Determination of the Concentration of Compound 1 for theReference Solution

[0223] A series of solutions with increasing concentrations of Compound1 in D₂O saline, pD 3.0 are prepared and the NMR relaxation rateconstants measured. Both T₁s and T₂s are measured because the drug, witha molecular weight of 1064.51, could lie outside the extreme motionalnarrowing limit for NMR, in which case, T₁s would be expected todecrease and T₂S would be expected to increase with a decrease inaggregation. See, K. Wüthrich, NMR OF PROTEINS AND NUCLEIC ACIDS, (1986)John Wiley Et Sons, New York. However, the data show an increase in bothT₁s and T₂S with decreasing aggregation, indicating that the drug isstill within the extreme motional narrowing limit under theseconditions. Table 1 summarizes the T₁ and T₂ values for the vinyl protonof Compound 1 which is located in the center of the molecule and isobserved to exhibit the largest changes. TABLE 1 NaCl Drug Conc. (mM) pD(mM) T₁ (secs) T₂ (secs) 160 3 150 0.83 0.13 80 3 150 1.20 0.38 40 3 1502.49 1.96 20 3 150 2.30 2.04 10 3 150 2.05 —

[0224] The data show that both the T₁ and T₂ values change as theconcentration of drug increases. Although one might be tempted toattribute a portion of the change in T₁ and T₂ to an expected increasein the viscosity of the solution with increasing concentration of drug(this was observed visually), such a change would be linear inproportion to the change in drug concentration. The data show relativelylittle change in T₁ and T₂ values between 10-40 mM of drug, a sharpdecrease in T₁ and T₂ between 40-80 mM of drug, and a smaller decreasein T₁ and T₂ between 80-160 mM of drug. The sharp decrease of T₁ and T₂is evidence that the critical micelle concentration of Compound 1 in D₂Osaline is between 40-80 mM. For this reason a concentration of 80 mM wasselected as the concentration of the drug for the reference solution.

[0225] C. Test Excipient: Citric Acid

[0226] A series of solutions containing 80 mM Compound 1 with increasingconcentrations of d₄-citrate in D₂O saline, pD 3.0, are prepared, andthe NMR relaxation rates are measured. The data obtained is summarizedin Table 2 and reported graphically in FIG. 2. TABLE 2 Drug Conc. NaClD₄-Citrate (mM) pD (mM) (mM) T₁ (secs) T₂ (secs) 80 3 150 100 1.96 0.8580 3 150 50 2.13 1.31 80 3 150 25 2.03 0.87 80 3 150 12.5 2.09 1.01 80 3150 6.25 1.24 0.69 80 3 150 3.13 1.69 0.67 80 3 150 1.56 1.65 0.59 80 3150 0.78 1.53 0.57 80 3 150 0 1.20 0.38

[0227] The data show that T₁ and T₂ values increase with increasingconcentration of d₄-citrate. This trend is indicative of decreasingaggregation with a maximal effect obatined at 50 mM d₄-citrate.Viscosity changes are discounted as having a minimal effect on T₁ and T₂values measured, because no visible changes in viscosity are noticed andit is not expected that addition of d₄-citrate to an aqueous solution inthese concentrations would substantially alter the viscosity.

[0228] The data show that a 50 mM concentration of d₄-citrate exhibitedrelaxation rates at least 25 percent slower than the relaxation rate ofthe reference solution containing the same concentration of the drug (alonger time in secs. for the measured T₁ and T₂ values is indicative ofa slower relaxation rate, which is indicative of reduced or loweredaggregation). Thus, citric acid at a 50 mM concentration is a suitableexcipient for decreasing aggregation of Compound 1 in solution.

[0229] D. Test Excipient: Citric Acid+Calcium Chloride

[0230] A series of solutions containing 80 mM Compound 1 and varyingconcentrations of CaCl₂ and d₄-citrate in D₂O saline, pD 3.0, areprepared, and the NMR relaxation rates are measured. The data obtainedis summarized in Table 3. TABLE 3 Drug Conc. NaCl D4-Citrate CaCl₂ (mM)pD (mM) (mM) (mM) T₁ (secs) 80 3 150 0.78 100 2.78 80 3 150 50 100 2.2680 3 150 5 100 2.14 80 3 150 5 50 1.94 80 3 150 5 25 1.89 80 3 150 512.5 1.51 80 3 150 5 6.25 1.47 80 3 150 5 3.13 1.45 80 3 150 0 0 1.2

[0231] As can be seen from the data, all but the two lowestconcentrations of CaCl₂ produce a change in the T₁ value (i.e., aslowing of the relaxation rate) which is greater than 25% as compared tothe relaxation rate of the reference solution containing the sameconcentration of Compound 1. This result indicates that combinations ofcalcium chloride and citric acid at these concentrations are sufficientto decrease aggregation of Compound 1 by at least about 25%.

[0232] The results obtained indicate the excipients at the indicatedconcentrations which produce a change in the T₁ value (i.e., a slowingof the relaxation rate) which is greater than 25% as compared to therelaxation rate of the reference solution containing the sameconcentration of drug. This result indicates that these excipients andcombinations of excipients are sufficient to decrease aggregation ofCompound 1 in solution by at least about 25%.

EXAMPLE 3

[0233] In vitro Rat Basophilic Leukemia (RBL) 2H₃Cell Studies

[0234] Histamine released by Compound 1, in the absence and presence ofadded excipients using Rat Basophil Leukemia (RBL) 2H3 cells isevaluated. RBL 2H3 is a rat mast cell line which stores preformedhistamine. RBL 2H3 cells (8×10⁴ cells) are seeded on a 96-well plateovernight and treated with of a formulation containing 160 mM ofCompound 1 and various concentrations of citric acid (0, 5, 10, 25 and50 mM) in distilled water at pH 3, for 30 minutes at 37° C. beforecollecting the cell-free media. The control, using the same cellcultures on the same day, involves exposing the cells to a formulationcontaining 160 mM of drug and 0 mM of citric acid in distilled water atpH 3. Histamine is quantified using the histamine assay (ELISA) kitcommercially available from Immunotech.

[0235] The data obtained is reported graphically in FIG. 2.

[0236]FIG. 2 shows the effects, in terms of percent ofpharmaceutically-induced histamine release, caused by the drug, versuscontrol, of the various concentrations of citric acid on the inhibitionof histamine release from RBL cells in vitro. FIG. 2 shows an inverserelationship between increasing concentrations of citric acid and thepercent histamine released from the RBL cells.

[0237]FIG. 2 also shows an inverse relationship between histaminerelease observed in RBL cells with increasing concentration of citricacid and Compound 1 aggregation, as indicated by the NMR relaxation rate(T₁), in solutions which contain corresponding concentrations of citricacid.

EXAMPLE 4

[0238] Human Blood Studies

[0239] Histamine release induced by Compound 1 is quantified in theabsence and presence of various excipients in peripheral (venous) bloodobtained from human donors. The blood is heparinized upon collection anddiluted 1:1 with saline buffer. Excipient solutions are prepared asaqueous solutions at pH 3 and pH 7 by solubilizing the appropriateamount of the specific excipient in water to prepare a solution with thedesired concentration of excipient. A sufficient amount of Compound 1 isthen added to each excipient solution to obtain a formulation containing40 mM of drug and the specified concentration of excipient. Formulationsare tested at both pH 3 and pH 7 to evaluate the effects of the drug inblood once the formulation is buffered by the blood.

[0240] Diluted blood is incubated with the prepared formulations for 5minutes at 37° C. before collecting cell-free medium. A control is runon the same day, using a formulation containing the same concentrationof the drug and no additional excipients. The amount of histamine in themedium is measured utilizing an immunoassay (ELISA) kit commerciallyavailable from Immunotech. The results obtained for each formulation arereported in Table 5 below in terms of the percent inhibition ofhistamine release at the indicated pH based on the control. TABLE 5 %Inhibition of Histamine Release Excipient Solution Conc. pH 3 pH 7glycine 12.5 mg/mL 75.2 ± 2.1  30.4 ± 2.5  EDTA-Na₂ 0.1% 14.1 ± 8.3 10.4 ± 5.6  glycine 12.5 mg/mL + EDTA-Na₂ 0.1% 45.4 ± 1.3  48.4 ± 1.7 glycine 12.5 mg/mL + citric acid 50 mM 44.3 ± 4.0  40.7 ± 0.7  citricacid 50 mM + EDTA-Na₂ 0.1% 37.9 ± 5.7  37.4 ± 1.6  citric acid 50 mM90.8 ± 14.6 3.5 ± 0.9 succinic acid 50 mM 77.5 ± 15.4  7.2 ± 14.6glucuronic acid 50 mM 66.9 ± 9.5  16.8 ± 8.5  L-tartaric acid 50 mM 65.1± 13.1 0 phosphoric acid 50 mM 60.4 ± 13.2 2.1 ± 8.8 acetic acid 50 mM59.9 ± 17.5 3.5 ± 0.6 maleic acid 50 mM 56.3 ± 7.9  0 bovine serumalbumin 10 mg/mL 39.8 ± 5.5  not tested lysine 12.5 mg/mL 25.8 ± 5.9  0D-aspartic acid 0.32 mg/mL 13.6 ± 7.7  0 arginine 12.5 mg/mL 0 30.4 ±1.8  magnesium sulphate 50 mM 0 9.5 ± 2.6

[0241] The results reported in Table 5 indicate that severalformulations suppress histamine release in human blood. The resultsdemonstrate that this method is useful for screening differentexcipients at different concentrations and under different pH conditionsto determine the appropriate excipients, concentrations and pH for agiven drug.

EXAMPLE 5

[0242] Formulations

[0243] Formulations containing a variety of drugs known or suspected topossess an adverse side-effect potential for histamine release in vivowhen administered as a rapid intravenous bolus or infusion are preparedaccording to the following procedure. The desired amount of excipient issolubilized in filtered water for injection or sodium chloride forinjection, pH is adjusted as necessary to obtain a solution having theindicated pH value, the solution is brought to volume with water orsodium chloride for injection to obtain the desired excipientconcentration, mixed well. The desired amount of Compound 1 is added,the formulation mixed, and then sterile filtered using 0.22-0.45 μm poresize membrane. The formulations are reported in Table 6. TABLE 6 Drug(conc.) pH Excipients A Compound 1 3 glycine (12.5 mg/mL) (10 mg/mL) BCompound 1 3 EDTA (0.1%)   (5 or 10 mg/mL) C Compound 1 3 glycine (12.5mg/mL) + EDTA (0.1%) (10 mg/mL) D Compound 1 3 glycine (12.5 mg/mL) +citric acid (50 mM) (10 mg/mL) E Compound 1 3 citric acid (50 mM) + EDTA(0.1%)   (2 or 10 mg/mL) F Compound 1 3 citric acid (50 mM) (10 mg/mL) GCompound 1 3 CaCl₂ (50 mM) (10 mg/mL) H Compound 1 3 glycine (12.5mg/mL) + citric acid (50 mM) + (10 mg/mL) EDTA (0.1%) I Compound 1 3glycine (12.5 mg/mL) + citric acid (50 mM) + (10 mg/mL) CaCl₂ (50 mM) Jmorphine sulphate 2.5-3.5 EDTA (0.1-0.2%) (8, 10 or 15 mg/mL) K morphinesulphate 2.5-3.5 citric acid (60 mM) (8, 10 or 15 mg/mL) L morphinesulphate 2.5-3.5 glycine (20 mg/mL) (8, 10 or 15 mg/mL) M morphinesulphate 2.5-3.5 CaCl₂ (40 mM) (8, 10 or 15 mg/mL) N morphine sulphate2.5-3.5 glycine (15 mg/mL) + EDTA (0.15%) (8, 10 or 15 mg/mL) O morphinesulphate 2.5-3.5 citric acid (40 mM) + EDTA (0.15%) (8, 10 or 15 mg/mL)P nalbuphrine HCl 3.5-3.7 EDTA (0.1-0.2%)  (10 or 20 mg/mL) Qnalbuphrine HCl 3.5-3.7 citric acid (60 mM)  (10 or 20 mg/mL) Rnalbuphrine HCl 3.5-3.7 glycine (20 mg/mL)  (10 or 20 mg/mL) Snalbuphrine HCl 3.5-3.7 glycine (5 mg/mL) + citric acid (20 mM) +  (10or 20 mg/mL) EDTA (0.1%) T oxymorphone HCl 3-5 citric acid (40 mM) +EDTA (0.2%)  (1 or 1.5 mg/mL) U oxymorphone HCl 3-5 citric acid (60 mM) (1 or 1.5 mg/mL) V remifentanil 3 glycine (15 mg/mL) (10 mg/mL) Wremifentanil 3 glycine (7 mg/mL) + citric acid (50 mM) + (10 mg/mL)CaCl₂ (50 mM) X mivacurium Cl 3.5-5   glycine (12.5 mg/mL)  (2 mg/mL) Ymivacurium Cl 3.5-5   EDTA (0.1%)  (2 mg/mL) Z mivacurium Cl 3.5-5  glycine (12.5 mg/mL) + EDTA (0.1%)  (2 mg/mL) AA mivacurium Cl 3.5-5  glycine (12.5 mg/mL) + citric acid (50 mM)  (2 mg/mL) BB mivacurium Cl3.5-5   citric acid (50 mM) + EDTA (0.1%)  (2 mg/mL) CC mivacurium Cl3.5-5   citric acid (50 mM)  (2 mg/mL) DD mivacurium Cl 3.5-5   CaCl₂(50 mM)  (2 mg/mL) EE mivacurium Cl 3.5-5   glycine (12.5 mg/mL) +citric acid (50 mM) +  (2 mg/mL) EDTA (0.1%) FF mivacurium Cl 3.5-5  glycine (12.5 mg/mL) + citric acid (50 mM) +  (2 mg/mL) CaCl₂ (50 mM) GGatracurium besylate 3.25-3.65 EDTA (0.1%) (10 mg/mL) HH atracuriumbesylate 3.25-3.65 glycine (12.5 mg/mL) + EDTA (0.1%) (10 mg/mL) IIatracurium besylate 3.25-3.65 citric acid (55 mM) (10 mg/mL) JJatracurium besylate 3.25-3.65 glycine (12.5 mg/mL) + citric acid (10mg/mL) (50 mM) + EDTA (0.1%) KK rocuronium Br 4 EDTA (0.15%)   (2 or 10mg/mL) LL rocuronium Br 4 citric acid (60 mM)   (2 or 10 mg/mL) MMrocuronium Br 4 citric acid (60 mM) + EDTA (0.15%)   (2 or 10 mg/mL) NNrapacuronium Br 3-5 citric acid (60 mM) (10 mg/mL)

EXAMPLE 6

[0244] In vivo Dog Screen

[0245] Using anesthetized beagle dogs, the formulations A-1 according toExample 5, are screened for their ability to inhibitpharmaceutically-induced histamine release in vivo. All studies areconducted in accordance with the USDA Animal Welfare Act and with strictadherence to the guidelines set forth by the Institutional Animal Careand Use Committee.

[0246] Adult male beagle dogs are anesthetized, intubated andartificially respired. The trachea is intubated under topical anesthesia(2% lidocaine) and ventilation is controlled to end-tidal CO₂concentrations of 25-30 mmHg. The animal is ventilated with a mixture ofoxygen and isoflurane to maintain a surgical level of anesthesiathroughout the experiment. Rectal temperature, end-tidal CO₂, andperipheral O₂ saturation (pulse oximetry) were monitored continuously. Acatheter is introduced into a femoral artery and connected externally toa pressure transducer. Blood pressure and heart rate are continuouslymonitored on a chart recorder.

[0247] In this experiment, histamine release is evaluated by changes inblood pressure (surrogate cardiovascular marker of histamine release) aswell as by measuring the concentrations of plasma histamine. The bloodpressure response to histamine release consists of a characteristicbrief fall at a latency of about 15-20 seconds from the time of bolusinjection. A concomitant tachycardic response is sometimes, but notalways, evident.

[0248] Blood samples are collected in chilled vials coated with EDTA 1min before injection and at the peak of the blood pressure response. Theplasma is extracted following standard procedures and stored immediatelyat −70° C. until analyzed. The plasma levels of histamine are determinedby immunoassay (ELISA) using a commercially available assay kit fromImmunotech.

[0249] Each animal serves as its own control: baseline (control)responses to incremental intravenous doses of drug (in sodium chloridefor injection, pH 3) are first obtained in each animal. At intervals of2-3 weeks, an identical dosing protocol is repeated in each animal witha formulation of the drug. The active is administered intravenously inincremental bolus doses at intervals of about 20 minutes. Controls areintermittently re-tested to guard against any drifts in the baselineresponses. The results of representative examples are reported in Tables7A-B below. TABLE 7 A Effects of Various Formulations in an AnesthetizedBeagle Dog (“Ethan”) Mean Arterial Plasma Histamine^(†) BloodPressure^(♦) Dose of Dose of Compound 1 (mg/kg): Compound 1 (mg/kg):Week Formulation 0.2 0.4 0.8 1.6 0.2 0.4 0.8 1.6 1 Control 6 90 846 1 9−34 3 A* 5 58 454 1042 1 −4 −13 −38 5 G* 21 113 585 975 5 −5 −7 −25 7Control 48 417 1125 −2 −2 −26 9 D* 4 65 180 707 2 0 −13 −21 11 Control56 269 1169 14 12 −31 13 E* 10 104 365 399 12 −5 −20 −29 15 A* 32 391125 1 2 −30 17 Control 33 385 1289 2 −7 −20

[0250] TABLE 7 B Effects of Various Formulations in an AnesthetizedBeagle Dog (“Dexter”) Mean Arterial Plasma Histamine^(†) BloodPressure^(♦) Dose Dose of of Compound 1 (mg/kg): Compound 1 (mg/kg):Week Formulation 0.2 0.4 0.8 0.2 0.4 0.8 1 Control 29 283 1191 22 −7 −343 A* 4.4 60 585 13 11 −15 5 C* 6 36 190 3 13 −25 7 Control 9 229 1269 1−8 −33 9 B* 1 25 181 3 4 −15 11 Control 7 69 531 −3 0 −51 13 D* 2 25 3670 4 −47 15 B* 1 14 394 0 −5 −32 17 Control 8 31 1243 4 9 −33

[0251] Tables 7A-B show the effects of several formulations in theanesthetized beagle dog screen. While, in some cases, the magnitude ofthe histamine-related depressor response to the drug does not appear tobe consistently affected by certain formulations, the plasma levels ofhistamine in each case is reduced, especially at the higher doses. Thisapparent discrepancy may be attributable to: 1) the selection of theseanimals is based on their ability to elicit a strong histamine-relatedresponse to the drug; and 2) repeated testing at short intervals mayhave depleted the stores of histamine in these animals, leading toincreased histamine receptor sensitivity. Tables 7A-B also display, insome cases, drifts in the control responses to rises in plasma histaminelevels and/or falls in mean arterial blood pressure (e.g., Dexter, week11). These drifts may be attributable to changes in the physicalcondition of the animal (e.g. bite wounds from other dogs, loss ofappetite and weight, etc.) leading to altered stores of histamine and/oraltered histamine receptor sensitivity. These examples demonstrate theneed for repeating experiments in animals which display more consistentcontrol responses. Also, longer rest periods between testing (3-4 weeks)would help replenish the lost histamine stores and retain histaminereceptor sensitivity. The latter regimen of testing was, therefore,successfully applied to the in vivo monkey studies described below.

EXAMPLE 7

[0252] In vivo Monkey Studies

[0253] Formulation A, B and E are evaluated (both for NMB andcardiovascular profiles) in anesthetized Rhesus monkeys. These studiesare conducted to ensure that the formulation improved thepharmaceutically-induced histamine release profile of Compound 1 withoutadversely affecting the primary neuromuscular blocking (i.e.,therapeutic) properties of the drug in a primate model. All studies areconducted in accordance with the USDA Animal Welfare Act and with strictadherence to the guidelines set forth by the Institutional Animal Careand Use Committee.

[0254] The neuromuscular blocking (NMB) properties and thehistamine-related cardiovascular effects of the drug formulated inFormulations A, B, and E according to Example 5, is evaluated inanesthetized Rhesus monkeys as follows: Formulation A, 1 monkey;Formulation B, 3 monkeys and Formulation E, 6 monkeys. Transient fallsin mean arterial blood pressure (MAP) are monitored as an index ofhistamine release. Twitch responses of the extensor digitorum of thefoot evoked by electrical stimulation of the peroneal nerve are used asan index of neuromuscular activity. NMB parameters measured or computedincluded: ED₉₅ (dose producing 95% suppression of twitch) as an index ofpotency; onset (time from injection to peak suppression of twitch) andduration of block (time from injection to 95% recovery of twitch).

[0255] The formulation of the drug is administered intravenously inincremental bolus doses (0.02-3.2 mg/kg) at intervals of about 30minutes. The first few doses (0.02-0.08 mg/kg) are administered toobtain data for constructing ED₉₅ log-probit curves. The next doseadministered is 0.8 mg/kg, after which the dose is successively doubleduntil a robust MAP response is elicited. Each animal served as its owncontrol.

[0256] Control (baseline) NMB and MAP responses are first obtained ineach animal. The control formulation includes 10 mg/mL of the drug and10 mM citric acid in saline at pH 3. At intervals (resting periods) of3-4 weeks, the effects of the drug in each formulation are re-evaluatedin each animal following an identical dosing regimen. The 3-4 week restperiod allows the animals to replenish their histamine stores and torecover from the minor surgery of the experiment. In addition, in thethree monkeys used in formulation B studies, control experiments arerepeated at an interval of 13 weeks (during which period otherformulations are evaluated at 3-4 week intervals) to determine theextent of drift in baseline (controls) NMB and MAP responses.

[0257] The results of the study are reported in Table 8. TABLE 8 TimeTreatment/Dose* Changes in Mean Arterial Sample of Compound 1 (mg/kg)Blood Pressure (mmHg) Formulation B Study Results (n = 3) Week 1  0.05  0 ± 1.0 Control 0.86 −12 ± 4.7 1.71 −22 ± 6.0 Week 5  0.05   4 ± 2.9Formulation B 0.77   2 ± 1.9 1.54 −11 ± 8.0 3.09  −11 ± 10.2 Week 130.04   1 ± 1.9 Control 0.86  −5 ± 3.6 1.71 −21 ± 5.0 Formulation E StudyResults (n = 6) Week 1  0.05   0 ± 0.5 Control 0.81  −7 ± 3.1 1.63 ‥±4.0 Week 5  0.05   3 ± 1.7 Formulation E 0.77  −3 ± 1.6 1.54  −9 ± 3.83.09 −16 ± 3.2

[0258] Table 8 indicates the changes in MAP elicited by formulations Band E versus the controls. Also provided in the table are the effects ofthe controls re-tested at the end of the formulation B study todetermine the fidelity of each response. As shown in the table, bothformulations B and E increased the mean dose of drug required to elicita comparable histamine-related depressor response by a factor ofapproximately 2 (range: 1- to 4-fold). The large standard errors inthese MAP values for formulation B are a function of the limited numberof study animals and the variability in the incidence and the magnitudeof the responses observed at a given dose. Re-testing of controls at theend of the formulation B study revealed some degree of variability inresponses at low doses, however, at higher doses, the responses remainedreproducible. The duration of action of Compound 1 appeared to besomewhat shortened by formulation E. None of the NMB properties of thedrug appeared to be adversely affected by either formulation. The dataindicate that formulation B and E suppressed thepharmaceutically-induced histamine release in the animals being treatedwith the drug, without adversely affecting the NMB properties of thedrug.

[0259] Formulation A (n=1) did not appear show a statisticallysignificant change in the effects of the drug on mean arterial bloodpressure in the single animal to which it was administered. However, theresults obtained are of limited reliability due to the study populationof 1.

[0260] An additional study is done in three monkeys to enable a directcomparison of the neuromuscular blocking potency of the drug in controlformulations versus the drug in formulation E. In this crossover study,several data points (yielding <99.9% block of the extensor digitorumtwitch) are obtained with low doses (0.02-0.08 mg/kg) of the controls.After a rest interval of 1 hour, during which nerve stimulation ishalted, an identical dosing protocol is followed with the drug informulation E. The results from this study revealed virtually identicalmean ED₉₅ values between the controls and formulation E (0.11±0.02mg/kg) indicating that the formulation did not adversely affect theneuromuscular blocking potency of the drug.

That which is claimed is:
 1. A method for preparing a pharmaceutical formulation containing a histamine releaser and a physiologically acceptable excipient, said method comprising combining a therapeutically effective amount of said histamine releaser with a concentration of the physiologically acceptable excipient; wherein said concentration of the physiologically acceptable excipient, when combined in an aqueous solution with the histamine releaser at or above critical micelle concentration, is sufficient to reduce aggregation of the histamine releaser in the aqueous solution by at least about 25 percent compared to aggregation of the histamine releaser in the aqueous solution containing substantially no physiologically acceptable excipient.
 2. The method according to claim 1, wherein the histamine releaser is a neuromuscular blocker.
 3. The method according to claim 1, wherein the histamine releaser is (Z)-2-chloro-1-{3-{(1R,2S)-6,7-dimethoxy-2-methyl-1-[(3,4,5-trimethoxyphenyl)methyl]-1,2,3,4-tetrahydro-2-isoquinolinio}propyl }-4-{3-[(1S,2R)-6,7-dimethoxy-2-methyl-1-(3,4,5-tri methoxyphenyl)-1,2,3,4-tetra hydro-2-isoquinolinio]propyl}-2-butenedioate dichloride or a pharmaceutically acceptable salt thereof.
 4. The method according to claim 1, wherein the physiologically acceptable excipient is selected from the group consisting of divalent inorganic salts, organic carboxylic acids, phosphoric acid, amino acids, chelating agents, albumins and combinations thereof.
 5. The method according to claim 1, wherein the physiologically acceptable excipient is selected from the group consisting of calcium chloride, sodium sulfate, magnesium sulfate, tartaric acid, maleic acid, acetic acid, citric acid, succinic acid, glucuronic acid, phosphoric acid, glycine, lysine, arginine, EDTA, bovine serum albumin, human serum albumin and combinations thereof.
 6. The method according to claim 1, wherein the concentration of the physiologically acceptable excipient is determined by the steps of: a) measuring aggregation of said histamine releaser in a reference solution consisting essentially of said histamine releaser in a concentration at or above the critical micelle concentration in the aqueous solution; b) measuring aggregation of said histamine releaser in a comparative solution consisting essentially of said histamine releaser and a pre-selected concentration of the physiologically acceptable excipient in the aqueous solution, wherein the concentration of said histamine releaser in the comparative solution is substantially the same as the concentration of said histamine releaser in the reference solution; c) optionally repeating step b) one or more times with a comparative solution having a different pre-selected concentration of the physiologically acceptable excipient; d) identifying a concentration of physiologically acceptable excipient that is sufficient to reduce aggregation of said histamine releaser in the comparative solution by at least about 25 percent compared to aggregation of said histamine releaser in the reference solution; wherein said identified concentration of step d) is the concentration of the physiologically acceptable excipient for combining with said histamine releaser to prepare the pharmaceutical formulation.
 7. A pharmaceutical formulation prepared according to claim
 1. 8. The pharmaceutical formulation according to claim 7, wherein said histamine releaser is a neuromuscular blocker.
 9. The pharmaceutical formulation according to claim 7, wherein said histamine releaser is (Z)-2-chloro-1-{3-{(1R,2S)-6,7-dimethoxy-2-methyl-1-[(3,4,5-trimethoxyphenyl)methyl]-1,2,3,4-tetrahydro-2-isoquinolinio}propyl }-4-{3-[(1S,2R)-6,7-dimethoxy-2-methyl-1-(3,4,5-trimethoxyphenyl)-1,2,3,4-tetrahydro-2-isoquinolinio]propyl}-2-butenedioate dichloride or a pharmaceutically acceptable salt thereof.
 10. The pharmaceutical formulation according to claim 7, wherein the physiologically acceptable excipient is selected from the group consisting of divalent inorganic salts, organic carboxylic acids, phosphoric acid, amino acids, chelating agents, albumins and combinations thereof.
 11. The pharmaceutical formulation according to claim 7, wherein the physiologically acceptable excipient is selected from the group consisting of calcium chloride, sodium sulfate, magnesium sulfate, tartaric acid, maleic acid, acetic acid, citric acid, succinic acid, glucuronic acid, phosphoric acid, glycine, lysine, arginine, EDTA, bovine serum albumin, human serum albumin and combinations thereof.
 12. The pharmaceutical formulation according to claim 7, wherein the physiologically acceptable excipient is a combination of any two or more excipients selected from the group consisting of glycine, EDTA, citric acid and calcium chloride.
 13. The pharmaceutical formulation according to claim 7, wherein the physiologically acceptable excipient is citric acid in a concentration of from about 15 mM to about 300 mM.
 14. The pharmaceutical formulation according to claim 7, wherein the physiologically acceptable excipient is EDTA in a concentration of from about 0.02 percent to about 1 percent.
 15. The pharmaceutical formulation according to claim 7, wherein the physiologically acceptable excipient is calcium chloride in a concentration of from about 15 mM to about 200 mM.
 16. The pharmaceutical formulation according to claim 7, wherein the physiologically acceptable excipient is a combination of citric acid in a concentration of from about 15 mM to about 100 mM and EDTA in a concentration of from about 0.02 percent to about 1 percent.
 17. The pharmaceutical formulation according to claim 7, wherein the physiologically acceptable excipient is a combination of citric acid in a concentration of from about 15 mM to about 100 mM and calcium chloride in a concentration of from about 25 mM to about 75 mM.
 18. The pharmaceutical formulation according to claim 7, wherein the physiologically acceptable excipient is glycine in a concentration of from about 10 mg/mL to about 100 mg/mL.
 19. The pharmaceutical formulation according to claim 7, wherein the physiologically acceptable excipient is a combination of glycine in a concentration of from about 10 mg/mL to about 100 mg/mL and EDTA in a concentration of from about 0.02 percent to about 1 percent.
 20. The pharmaceutical formulation according to claim 7, wherein the physiologically acceptable excipient is a combination of glycine in a concentration of from about 10 mg/mL to about 100 mg/mL and citric acid in a concentration of from about 15 mM to about 100 mM.
 21. The pharmaceutical formulation according to claim 7, wherein the physiologically acceptable excipient is a combination of glycine in a concentration of from about 10 mg/mL to about 100 mg/mL, citric acid in a concentration of from about 15 mM to about 100 mM, and EDTA in a concentration of from about 0.02 percent to about 1 percent.
 22. The pharmaceutical formulation according to claim 9, wherein the physiologically acceptable excipient is citric acid in a concentration of about 50 mM.
 23. The pharmaceutical formulation according to claim 9, wherein the physiologically acceptable excipient is EDTA in a concentration of about 0.1 percent.
 24. The pharmaceutical formulation according to claim 9, wherein the physiologically acceptable excipient is calcium chloride in a concentration of about 50 mM.
 25. The pharmaceutical formulation according to claim 9, wherein the physiologically acceptable excipient is a combination of citric acid in a concentration of about 50 mM and EDTA in a concentration of about 0.1 percent.
 26. The pharmaceutical formulation according to claim 9, wherein the physiologically acceptable excipient is a combination of citric acid in a concentration of about 50 mM and calcium chloride in a concentration of about 50 mM.
 27. The pharmaceutical formulation according to claim 9, wherein the physiologically acceptable excipient is glycine in a concentration of about 12.5 mg/mL.
 28. The pharmaceutical formulation according to claim 9, wherein the physiologically acceptable excipient is a combination of glycine in a concentration of about 12.5 mg/mL and EDTA in a concentration of about 0.1 percent.
 29. The pharmaceutical formulation according to claim 9, wherein the physiologically acceptable excipient is a combination of glycine in a concentration of about 12.5 mg/mL and citric acid in a concentration of about 50 mM.
 30. The pharmaceutical formulation according to claim 9, wherein the physiologically acceptable excipient is a combination of glycine in a concentration of about 12.5 mg/mL, citric acid in a concentration of about 50 mM, and EDTA in a concentration of about 0.1 percent.
 31. The pharmaceutical formulation according to claim 7, further comprising a physiologically acceptable diluent.
 32. The pharmaceutical formulation according to claim 7, wherein said formulation has a pH of from about 2 to about
 8. 33. A method of suppressing pharmaceutically-induced histamine release in an animal being treated with the histamine releaser, said method comprising administering to said animal the pharmaceutical formulation according to claim
 7. 34. The method according to claim 33, wherein said step of administering the pharmaceutical formulation comprises intravenously administering the pharmaceutical formulation.
 35. A method for preventing cardiovascular and respiratory effects mediated by pharmaceutically-induced histamine release in an animal being treated with the histamine releaser, said method comprising administering to said animal the pharmaceutical formulation according to claim
 7. 36. The method according to claim 35, wherein said cardiovascular and respiratory effects mediated by pharmaceutically-induced histamine release are selected from the group consisting of flushing, hypotension, tachycardia, bronchoconstriction, anaphylactoid reactions and anaphylactic shock, and combinations of any two or more thereof.
 37. A method for determining a concentration of a physiologically acceptable excipient that is sufficient to suppress pharmaceutically-induced histamine release in an animal being treated with a histamine releaser, said method comprising the steps of: a) measuring aggregation of said histamine releaser in a reference solution consisting essentially of said histamine releaser in a concentration at or above the critical micelle concentration in an aqueous solution; b) measuring aggregation of said histamine releaser in a comparative solution consisting essentially of said histamine releaser and a pre-selected concentration of the physiologically acceptable excipient in the aqueous solution, wherein the concentration of said histamine releaser in the comparative solution is substantially the same as the concentration of said histamine releaser in the reference solution; c) optionally repeating step b) one or more times with a comparative solution having a different pre-selected concentration of the physiologically acceptable excipient; d) identifying a concentration of physiologically acceptable excipient that is sufficient to reduce aggregation of said histamine releaser in the comparative solution by at least about 25 percent compared to aggregation of said histamine releaser in the reference solution; wherein said identified concentration of step d) is the concentration of the physiologically acceptable excipient that is sufficient to suppress pharmaceutically-induced histamine release in an animal being treated with a histamine releaser.
 38. The method according to claim 37, wherein said step a) and said step b) of measuring aggregation comprise measuring NMR relaxation rates of the histamine releaser in the reference solution and the comparative solution, and said step d) of identifying a concentration of physiologically acceptable excipient comprises identifying a concentration of physiologically acceptable excipient sufficient to slow said measured NMR relaxation rates of the histamine releaser in the comparative solution by at least about 25 percent compared to said measured NMR relaxation rates of the histamine releaser in the reference solution.
 39. A method for preparing a pharmaceutical formulation containing a histamine releaser and a physiologically acceptable excipient, said method comprising combining a therapeutically effective amount of the histamine releaser with a physiologically acceptable excipient wherein the physiologically acceptable excipient is present in a concentration determined according to the method of claim
 37. 40. A pharmaceutical formulation prepared according to claim
 39. 41. The pharmaceutical formulation according to claim 40, wherein said histamine releaser is a neuromuscular blocker.
 42. The pharmaceutical formulation according to claim 40, wherein said histamine releaser is (Z)-2-chloro-1-{3-{(1R,2S)-6,7-dimethoxy-2-methyl-1-[(3,4,5-trimethoxyphenyl)methyl]-1,2,3,4-tetrahydro-2-isoquinolinio}propyl}-4-{3-[(1S,2R)-6,7-di methoxy-2-methyl-1-(3,4,5-trimethoxyphenyl)-1,2,3,4-tetra hydro-2-isoquinolinio]propyl}-2-butenedioate dichloride or a pharmaceutically acceptable salt thereof.
 43. The pharmaceutical formulation according to claim 40, wherein said physiologically acceptable excipient is selected from the group consisting of divalent inorganic salts, organic carboxylic acids, phosphoric acid, amino acids, chelating agents, albumins and combinations thereof.
 44. The pharmaceutical formulation according to claim 40, wherein said physiologically acceptable excipient is selected from the group consisting of calcium chloride, sodium sulfate, magnesium sulfate, tartaric acid, maleic acid, acetic acid, citric acid, succinic acid, glucuronic acid, phosphoric acid, glycine, lysine, arginine, EDTA, bovine serum albumin, human serum albumin and combinations thereof.
 45. The pharmaceutical formulation according to claim 40 further comprising a physiologically acceptable diluent.
 46. A method for suppressing pharmaceutically-induced histamine release in an animal being treated with the histamine releaser, said method comprising administering to said animal the pharmaceutical formulation according to claim
 40. 47. A method for preparing a pharmaceutical formulation containing a histamine releaser and a physiologically acceptable excipient, said method comprising combining a therapeutically effective amount of the histamine releaser with a concentration of physiologically acceptable excipient, wherein the concentration of the physiologically acceptable excipient is determined by a method comprising the steps of: a) measuring histamine release from a histamine-containing biological sample in a reference mixture consisting essentially of: i) the histamine-containing biological sample in a medium and ii) an aqueous solution of the histamine releaser at a concentration sufficient to cause histamine release from the histamine-containing biological sample; b) measuring histamine release from the histamine-containing biological sample in a comparative mixture consisting essentially of: i) the histamine-containing biological sample in medium and ii) an aqueous solution of the histamine releaser and a pre-selected concentration of the physiologically acceptable excipient,  wherein the histamine releaser in the comparative mixture is present in a concentration which is substantially the same as the concentration of histamine releaser in the reference mixture of step a); c) optionally repeating step b) one or more times with a comparative mixture having a different pre-selected concentration of the physiologically acceptable excipient; and d) identifying a concentration of the physiologically acceptable excipient sufficient to reduce histamine release from the histamine-containing biological sample in the comparative mixture by at least about 10 percent compared to histamine release from the histamine-containing biological sample in the reference mixture; wherein said identified concentration of step d) is the concentration of said physiologically acceptable excipient for combining with said histamine releaser to prepare the pharmaceutical formulation.
 48. A pharmaceutical formulation prepared according to claim
 47. 49. The pharmaceutical formulation of claim 48 further comprising a physiologically acceptable diluent.
 50. A method for suppressing pharmaceutically-induced histamine release in an animal being treated with the histamine releaser, said method comprising administering to said animal the pharmaceutical formulation according to claim
 48. 51. A method for preventing cardiovascular and respiratory effects mediated by pharmaceutically-induced histamine release in an animal being treated with the histamine releaser, said method comprising administering to said animal the pharmaceutical formulation according to claim 48
 52. A kit for preparing a pharmaceutical formulation of the histamine releaser, said kit comprising: a) a physiologically acceptable excipient, and b) instructions for preparing the pharmaceutical formulation according to claim
 1. 53. The kit according to claim 52 wherein the physiologically acceptable excipient is selected from the group consisting of divalent inorganic salts, organic carboxylic acids, phosphoric acid, amino acids, chelating agents, albumins and combinations thereof.
 54. The kit according to claim 53, wherein the physiologically acceptable excipient is selected from the group consisting of calcium chloride, sodium sulfate, magnesium sulfate, tartaric acid, maleic acid, acetic acid, citric acid, succinic acid, glucuronic acid, phosphoric acid, glycine, lysine, arginine, EDTA, bovine serum albumin, human serum albumin and combinations thereof.
 55. The kit according to claim 52 further comprising a physiologically acceptable diluent and instructions for combining the therapeutically effective amount of said histamine releaser with the physiologically acceptable excipient in the physiologically acceptable diluent.
 56. The kit according to claim 52, wherein said instructions further comprise instructions for adjusting pH of the pharmaceutical formulation to from about 2 to about
 8. 57. A pharmaceutical formulation comprising a therapeutically effective amount of (Z)-2-chloro-1-{3-{(1R,25)-6,7-dimethoxy-2-methyl-1-[(3,4,5-trimethoxyphenyl)methyl]-1,2,3,4-tetrahydro-2-isoquinolinio}propyl}-4-{3-[(1S,2R)-6,7-dimethoxy-2-methyl-1-(3,4,5-tri methoxyphenyl)-1,2,3,4-tetrahydro-2-isoquinolinio]propyl}-2-butenedioate dichloride or a pharmaceutically acceptable salt thereof; an excipient selected from the group consisting of glycine in a concentration of from about 10 mg/mL to about 30 mg/mL, citric acid in a concentration of from about 25 mM to about 75 mM, EDTA in a concentration of from about 0.1% to about 0.5%, calcium chloride in a concentration of from about 25 mM to about 75 mM, and combinations thereof; and a physiologically acceptable diluent, wherein said pharmaceutical formulation is suitable for intravenous administration.
 58. A pharmaceutical formulation comprising a histamine releaser and a physiologically acceptable excipient wherein the concentration of the physiologically acceptable excipient is sufficient to suppress pharmaceutically induced histamine release.
 59. Use of a pharmaceutical formulation according to any of claims 7, 40, 48, 57, or 58 for the manufacture of a medicament for suppressing pharmaceutically-induced histamine release in an animal being treated with the histamine releaser.
 60. The use according to claim 59, wherein the medicament is administered intravenously.
 61. Use of a pharmaceutical formulation according to any of claims 7, 40, 48, 57, or 58 for the manufacture of a medicament for preventing cardiovascular and respiratory effects mediated by pharmaceutically-induced histamine release in an animal being treated with the histamine releaser.
 62. The use according to claim 61 wherein the cardiovascular and respiratory effects mediated by pharmaceutically-induced histamine release are selected from the group consisting of flushing, hypotension, tachycardia, bronchoconstriction, anaphylactoid reactions and anaphylactic shock, and combinations of any two or more thereof. 